To be directed by Tony Award-winner Anthony Page, the show is set to begin preview performances April 10 at Studio 54, with an opening night planned for April 30, the Roundabout Theatre Company announced.
Irwin will play Vladimir and Lane will portray Estragon in the limited engagement production.
Bob Fosse's "Dancin,'" previously announced for May, will now be part of the 2009-2010 Roundabout season, the company said.
Additional cast members and the design team for "Waiting for Godot" will be announced shortly.
Page recently directed Irwin in his Tony-winning performance in "Who's Afraid of Virginia Woolf?"
Lane has starred in "The Man Who Came to Dinner," "The Producers" and "The Odd Couple."