Chamberlin was born in Baltimore, Maryland, and raised in Moorestown Township, New Jersey, moving there as a nine-year-old. Chamberlin graduated from Rutgers University's Mason Gross School of the Arts with a Bachelor of Fine Arts in acting.
Chamberlin has been nominated for Drama Desk and Tony Awards for Dirty Blonde (as Charlie), Seussical (as Horton), and The Addams Family (as Uncle Fester). Additional Broadway theatre credits include My Favorite Year, Triumph of Love, Abe Lincoln in Illinois, Chicago, and The Ritz. He also appeared in the 1999 gay-themed movie Trick.
He also appeared in Die Hard with a Vengeance as an enthusiastic NYPD bomb defusal expert. In Lucky Number Slevin, he again had a supporting role as a New York police officer. Chamberlin's most recent work includes the role of Aron Malsky in the NBC primetime series Heroes.
Chamberlin previously appeared as Uncle Fester in the musical The Addams Family, a role for which he won a Broadway.com Audience Award for Favorite Performance by a Featured Actor In a Broadway Musical. Brad Oscar replaced him in the role of Uncle Fester on March 8, 2011.
In the Summer 2013 Chamberlin starred along with Ross Lynch and Maia Michell in the Disney Channel Original Movie Teen Beach Movie as Dr. Fusion.