We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ubiquitin-Activated Interaction Traps) are E3-ubiquitin fusion proteins, and in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. UBAITs were made for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, substrate trapping occurred in vitro either through an E3 thioester-linked lariat intermediate or an E2 thioester intermediate, and both WT and active site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells.