Tim Conway was famous on The Carol Burnett Show for ad-libbing and cracking everyone up. In this skit, one that had many manifestations, the family of Mama (Vicki L) was playing a game. Conway was making all of that up as he went along. They were all trying not to laugh, so when they were trying to get back on track Ms Lawrence said "Are you sure that little assholes done?"
I guess you do have to know the show, and the history behind it all. Youtube is rife (rife, I tell you) with takes from the show where Conway went off on a tangent.
It was some years ago too. Many of us grew up with The Carol Burnett show, so it might be funny to someone other than me.
Out of context, I guess it wouldn't be funny.