8 traces the story of California's gay marriage ban Proposition 8 and the fight by a network of activists and lawyers to have it overturned.
Pitt will star in the March 3 staging as the US district chief judge who found Proposition 8 unconstitutional.
Clooney and Martin Sheen portray David Boies and Theodore B Olson, the two high-profile lawyers who worked to overturn the gay marriage ban.
Glee's Jane Lynch, Matthew Morrison, George Takei, Matt Bomer and Jesse Tyler Ferguson will also appear in the one-off version of 8.
8 was written by Dustin Lance Black, the Oscar-winning screenwriter of Gus Van Sant's 2008 film Milk.
Proceeds from the event will benefit the American Foundation for Equal Rights.
The fundraising staging of 8 takes place Saturday, March 3 at Wilshire Ebell Theatre and will be streamed live on YouTube.
> George Clooney: 'I don't give a s**t if people think I'm gay'
> Brad Pitt: 'Gay marriage is constitutional right'