Perabo's parents named her after Piper Laurie. Her father, George William Perabo, is Portuguese and her mother, Mary Charlotte Ulland, is Norwegian American. Her brothers, Noah and Adam, are also actors. Perabo graduated summa cum laude from Ohio University (in Athens, Ohio) in 1998 with a Bachelor of Fine Arts in Acting. After University, she moved to New York and was cast in her first movie — a short film called Single Spaced, where she played The Dame. Her first big role came after, where she played the girlfriend of a wannabe rapper in Whiteboyz. She also did another short film, Knuckleface Jones. 2000 was a big year for her: she was cast in The Adventures of Rocky and Bullwinkle as one of the lead characters, FBI agent Karen Sympathy. The break-out role that got wide recognition and shot her into the public eye was in Coyote Ugly, where she played Violet Sanford aka 'Jersey', who moves to New York to pursue her dream of becoming a songwriter. She had to take lessons in voice, guitar, piano and bartending to prepare for the role. Despite the movie not doing as well as it could have at the box office, she won an MTV Movie Award for Best Music Moment for "One Way or Another".

In 2001, she starred in an independent Canadian movie called Lost and Delirious, playing a boarding school student who falls in love with a girl. The next year she starred as a French exchange student in Slap Her... She's French, which was shelved in North America for two years, then released under the new title She Gets What She Wants. The film was released under its original title in Europe. In 2003, she had a small role as the eldest daughter in Cheaper by the Dozen (2003) which she reprised in its 2005 sequel.