Eric Idle will be joined by Michael Palin, Terry Jones and Terry Gilliam, as well as Carol Cleveland and Neil Innes, to perform Not The Messiah (He's A Very Naughty Boy).
However, fifth surviving Python John Cleese is unable to attend because of prior commitments, The Daily Telegraph reports.
Of the new piece, Idle said: "It ranges in reference from Handel, through a naughty Mozart duet, to the Festival Of Nine Carols, Bob Dylan, and the classic finale 'Always Look On The Bright Side of Life'.
"Michael Palin is going to appear in drag for the first time in several years - in public anyway. Only by the scruff of their necks have I got them all performing again. Some of the other Pythons go through wives rather quickly."
The performance, to take place at the Royal Albert Hall on October 23, will be the first show by the four Pythons since they appeared at the Concert For George tribute to late Beatle George Harrison in November 2002.