Lotte Verbeek was educated at Gymnasium Collegium Marianum in Venlo and graduated in 2008 at the Theaterschool in Amsterdam. From 1999, Verbeek attended the Dance Academy in Arnhem and the Amsterdam Academy of Jazz/Musical Theatre and Dance, where she graduated in 2006. During her studies she worked as a dancer and model for photographer Erwin Olaf.
After completing high school, Verbeek started her acting career performing lead roles in Dutch movies such as Moes (2006) and LEFT (2007), in which she plays five double roles.
In 2009 she won the Leopard for best actress at the Locarno International Film Festival for her role of Anne in Nothing Personal, Dutch movie directed by Urszula Antoniak. For the same part she was also the recipient of the best actress award at the International Film Festival of Marrakech 2009 and was nominated as best actress for the Golden Calf at the Netherlands Film Festival.
In 2010 Verbeek received the Shooting Stars Award, the annual acting award for up-and-coming actors by European Film Promotion, at the Berlin International Film Festival. In the same year she was cast in Le Ragazze dello Swing (The Swing Girls), an award-winning Italian mini-series based on the Trio Lescano story. For her main role as Judith Leschan she received a Golden Nymph award for best actress alongside Andrea Osvart and Elise Schaap at Monte-Carlo Television Festival 2011.
In 2011, she began playing Giulia Farnese in the Showtime period drama The Borgias, alongside Jeremy Irons in the role of Pope Alexander VI. Created by Neil Jordan and shot in Budapest, the series has been renewed for a third season in 2013.
In 2012 Verbeek played a double role of two twins, Therese and Angelique, in the erotic thriller Suspension of Disbelief, written and directed by Mike Figgis and screened at Rome Film Festival.
As stage actress, Verbeek was in the production of Maria Magdalena by Wayne Traub, which toured Europe in 2009.
Verbeek speaks Dutch, English, French, German and Italian. In addition to her drama and dance studies, she followed many singing lessons and workshops. Even though she can sing in the soprano vocal range, in Le Ragazze dello Swing the songs are performed by The Blue Dolls.