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 the same year. Prepon co-starred with Bryan Greenberg as Hannah Daniels in the ABC drama October Road, which debuted March 15, 2007. The show was canceled by ABC on May 12, 2008, despite strong ratings and a grassroots campaign from the show's fanbase to keep the show alive. In April 2007, she starred in a dramatic short film Once Upon a Time. She also appeared in the 2005 Oxygen romantic comedy Romancing the Bride. She also was the original voice for Hayley Smith on the Fox animated sitcom American Dad! and voiced her in an unaired pilot.  She appeared as Lauren, the long-lost sister of Mary Shannon (Mary McCormack) in the USA Network mystery drama In Plain Sight in the episode "A Frond in Need". On February 5, 2010, it was announced that Prepon signed on to star in Awkward Situations for Men, a pilot on ABC. In 2009, Prepon appeared in the CBS sitcom How I Met Your Mother as Karen, one of the girlfriends of Ted Mosby (Josh Radnor), in the episode "Sorry, Bro". She reprised her role in the episodes "The Front Porch" in 2009 and "Say Cheese" in 2010. She also appeared in the Fox medical drama House M. D. in the episode "Private Lives".