Larter landed her first professional roles in 1997 when she appeared in several television programs. She appeared in an episode of the Brooke Shields television series Suddenly Susan, and the short-lived series Chicago Sons. These roles were followed by a number of other appearances on Dawson’s Creek,Chicago Hope, and Just Shoot Me!

In 1999, Larter began her film career with an appearance in Varsity Blues, which re-united her with Dawson’s Creek star Van Der Beek and close friend Amy Smart. Smart had persuaded Larter to audition for the movie, in which she played Darcy Sears, a love interest for one of the main characters. Varsity Blues drew a domestic box office gross of $53 million.That year she made appearances in the teen comedies Giving It Up and Drive Me Crazy. Larter also starred in the horror remake House on Haunted Hill which was made for around $20 million. The movie was panned by critics, but grossed $15 million on its opening weekend and went on to earn over $40 million overall.

In 2000, Larter starred as one of the main characters, Clear Rivers, in the teen supernatural horror film Final Destination. Also starring Devon Sawa and Kerr Smith, the movie’s premise followed several teenagers who survive a plane crash but are stalked and killed by death itself. Final Destination made $112 million by the end of its theatrical run. The following year, she appeared in the comedy Legally Blonde with Reese Witherspoon. She played Brooke Taylor Windham, a widow accused of her husband’s murder. The film earned the top spot with $20 million in its opening weekend and ended up grossing $141 million worldwide.With $96 million of it total gross deriving from domestic markets, Legally Blonde is Larter’s highest grossing film domestically.

Larter next appeared as Zerelda Mimms in the western comedy American Outlaws Directed by Les Mayfield and co-starring Colin Farrell and Scott Caan, the movie was poorly received by criticsand at the box office making $13 million at the end of its theatrical run She also starred in Kevin Smith’s Jay and Silent Bob Strike Back. That year, Larter appeared on the cover of Maxim magazine and performed in the stage play The Vagina Monologues in New York Cityn Spring 2002, Larter moved from Los Angeles to New York. “I was too young and impressionable to handle the pressures of L.A.” Larter recalled in an interview, “I’m a woman now. I am no longer the little girl who could be easily influ­enced.”

Her first project there was to reprise her role as Clear Rivers in the sequel to Final Destination entitled Final Destination 2. In an interview with IGN, Larter explained her return to the franchise: “When New Line asked me to come back, I thought it was great. They showed me the script and let me have some input, and it was really terrific.”The film opened at number 2 with $16 million to mixed critical reception.A year later, Larter served as an associate producer of and starred in the thriller Three Way. In 2005, Larter appeared in the independent political thriller, Confess, and had a role in the romantic comedyA Lot Like Love alongside Amanda Peet and Ashton Kutcher.

Larter moved back to Los Angeles in 2005.Her first audition was for the NBC science fiction drama television seriesHeroes Larter played the characters of Niki Sanders, who suffered from DID, and Tracy Strauss on the show created byTim Kring. Larter’s initial character Niki Sanders, was a wife, mother, and a former internet stripper from Las Vegas who exhibits superhuman strength and alternate personalities who go by the names of Jessica and Niki. “Ali read for the part and just owned it from the second she walked in.”, Kring said to The Chicago Tribune, “It was a very impressive audition.”The series collected a number of accolades in its first season including a People’s Choice Award and nominations from the Primetime Emmy Awards and Golden Globes. As of the third season, Larter began to play a new character, Tracy Strauss, who possessed the ability to freeze objects; and later, turn her body into water. Larter ultimately decided against starring in the sequel to House on Haunted Hill stating, “I feel so lucky with the things I am doing right now, and that’s so long ago for me.”

In 2007, Larter starred in Marigold as the title character alongside Salman Khan. The movie, released in August 2007, was about an American actress (Larter) who goes to India and gets caught up in the exotic world of Bollywood. Filming took place in North India and London, commencing in June 2004. In an interview with the BBC, Larter explained how she came about the role of Marigold and expressed why she wanted to star in the film: “I was living in (director) Willard Carroll’s guesthouse when he gave me the script. He had written this really strong female character and for me, it was an opportunity to overcome my fear of singing and dancing because I have no professional training. Also, I would get to live in another country for couple of months. With Heroes I didn’t think of it as that huge sci-fi series and it was the same forMarigold. I really focused on the character and loved this journey she went on and the experiences she had.” She was paid a seven-figure salary for the role of Marigold.

It was this year that she was cast alongside Milla Jovovich in the horror film Resident Evil: Extinction, portraying the character of Claire Redfield. Her role sent her to Mexicali, Mexico for filming from May to late July, and required her hair to be dyed a light red. Larter explained her character Claire: “She became the leader of this convoy. She’s incredibly strong, patient. I think she serves a role for everyone within this convoy, let it be a mother to someone, a buddy, a best friend.” Larter also attended the 2007 Comic Con International, her second appearance at the event, to promote the movie. The movie was released in theaters on September 21, 2007 and made a total of $147 million worldwide. She appeared with Hayes McArthur in the comedy Homo Erectus the same year. She also appeared as Evelyn Garland in the biographical film Crazy, based on guitarist Hank Garland. In an interview for Resident Evil: Extinction, Larter had expressed interest in producing films in the future, saying, “I definitely have many ideas and different avenues that I want to take as my career goes on.”

In April 2009, Larter starred opposite Beyoncé Knowles and Idris Elba in the Screen Gems-produced thriller Obsessed.The film follows an office executive (Elba) whose marriage to Knowles’ character is threatened by the aggressive interests of a co-worker, portrayed by Larter. In an interview with Glam, Larter said that she “was excited to get the chance to play a femme fatale. I love playing women that are dark and vulnerable and sort of filled with a little bit of crazy emotion.” Derek Malcolm of The London Evening Standard said the movie was a “dim reworking of Fatal Attraction” and noted, “Larter as the pathological minx is the best thing about it.” Obsessed suffered critically but eventually opened at #1 in its opening weekend with $28 million  and with a total domestic gross of $68 million, became Larter’s second highest grossing film in North-America. It resulted in Larter’s third Teen Choice Awardnomination and an MTV Movie Award for Best Fight with Beyonce.

Larter reprised her role as Claire Redfield in Resident Evil: Afterlife, which was filmed in 3D and directed by Paul W.S. Anderson. She attended WonderCon and Comic Con to promote the movie. In an interview withJoBlo.com, Larter talked about her role in the film: “I guess people liked me as her…I’m excited that they brought me back. I love working with Milla, and it’s also very excited to have Paul back directing this. To work with the man who really created this world and this vision was what excited me about joining this next installment.”The movie was an international success, earning $296 million worldwide and becoming Larter’s highest grossing picture to date. As of August 2011, Larter’s films have grossed a total of $552 million domestically.

Recently, Larter appeared as the title character in a short film for Absolut Vodka entitled Lemon Drop. Larter is linked to the role of Col. Virginia Lake in the big-screen adaptation of UFO, which stars Joshua Jackson, and the role of Cheryl in a feature version of the Sundance short film, The Rambler directed by Calvin Reeder. While at WonderCon, Larter commented on Heroes’ possible fifth season. “I think that we’ll be back…I think that there are still stories that need to be told.” NBC announced the show’s cancellation on May 14, 2010.

Larter portrayed the lead role in a pilot for a series written by Josh Friedman entitled, “The Asset”. The show, which was expected to air on Fox, was not picked up for a series commission.However, Larter gained a role in TNT’s drama Legends as Crystal Quest, the female lead alongside Sean Bean. Her character plays an operative with the FBI’s Deep Cover Operations who has a history with Bean’s character. On May 8, 2013, the series was picked up for 10 episodes to air in 2014.