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Prepon appeared in plays such as A Woman of Property and Ascension Day while in New York. In 1996, she studied drama with acting teacher Caroline Thomas at Thomas’ Total Theatre Lab. Later, in September 1997, she premiered on a Levi Strauss-developed show called They Go On, an Internet-based soap opera.

Prepon starred as Donna Pinciotti on the Fox sitcom That ’70s Show from 1998 to 2006 and co-starred in the romantic comedy Slackers. In 2001, she made her film debut in Steve Hanft’s musical comedy-drama Southlander with Beck, Beth Orton and Hank Williams III, where she played Seven=Five, a young TV telepathic. In 2004, Prepon worked on the independent drama film The Pornographer: A Love Story.
Laura had a cameo voice appearance as a United Nations Space Command marine in Halo 2, which was released in 2004.

Her first notable film role came in 2004 with the dark drama Lightning Bug. In 2005, Prepon co-starred with Misha Collins in the psychological thriller Karla, based on the true story of Paul Bernardo and his wife Karla Homolka, a Canadian couple who kidnapped, sexually abused, and murdered three girls, marking a contrast to her usual lighthearted roles. She was also an executive producer of E! Hollywood Hold’em in the same year.

Prepon co-starred in the ABC drama October Road, which debuted March 15, 2007. The show was canceled by ABC on May 12, 2008. In April 2007, she starred in a dramatic short film Once Upon a Time. She also appeared in the 2005 Oxygen original romantic comedy Romancing the Bride. She give her voice for Hayley Smith on the Fox animated sitcom American Dad!.

She play various roles include In Plain Sight, How I Met Your Mother, House M.D., Medium, Castle and more. In February 2011, she was Chelsea Newman on the NBC sitcom Are You There, Chelsea?, based on Chelsea Handler’s 2008 book Are You There, Vodka? It’s Me, Chelsea. NBC cancelled the show on May 11, 2012.

In 2016, Prepon appeared in the psychological thriller The Girl on the Train and in 2017, she played Charlotte opposite Sam Elliott in The Hero.

Since 2013, Laura as Alex Vause in the Netflix original comedy-drama series Orange Is the New Black and also directed some episodes on season 5 and season 6.