The 'Hollywood Bites Back!' benefit will be held at LA Live's Nokia Theatre on April 16 to raise awareness and money for the non-profit group Malaria No More.
Other stars announced as appearing at the one-night-only event are Next Three Days actress Elizabeth Banks, Office writer-star BJ Novak and David Arquette.
"I am honoured to have been asked to take part in this important event," O'Brien said in an official statement. "I just hope that we can find a better cure for malaria than comedy."
Malaria No More was founded in 2006 by Fox chairman Peter Chernin and UN Secretary-General's Special Envoy for Malaria Ray Chambers to help eradicate the disease in Africa by 2015.
O'Brien recently joined fellow comics Jon Stewart and Sarah Silverman for a televised tribute to the late comedian Greg Giraldo.