How do I play against GLaDOS in Poker Night 2?

I don't think that it's possible to ever play against GLaDOS. What you probably heard is that it's possible to win an item from her, which you get if you beat all the bounty challenges after getting the items from the other four. However, she only deals and insults you still.

you can get Glados to enter the competition, if I remember right, if you get all the other player's 'Bounties' Glados joins the competition. (If you're finding it hard, i find that Omaha makes the game more easy. Oh yeah, and the reason that you are finding it hard to play the game, is because the NPCs can now detect bluffs much better than the previous players.)