The small ubiquitin-related modifier (SUMO) is a protein of ~10kDa that is covalently conjugated to its substrate proteins in an enzymatic process called sumoylation. This posttranslational modification is an essential regulatory mechanism that plays crucial roles in many cellular pathways. It allows rapid adaptation to environmental changes by switching protein functions due to alternate complex assemblies, changes in intracellular localization, enzymatic activity, or stability. SUMO conjugation is executed by the hierarchical action of E1, E2, and E3 enzymes. Both E2 and E3 enzymes contribute to substrate specificity but with E3 ligases being the more important for this. E1 and E2 activities are essential for all sumoylation reactions but usually-with a few exceptions-modify substrates only inefficiently. Hence, most substrates require the additional action of an E3 ligase or a cofactor. Here, we describe methods to distinguish a bona fide E3 ligase from a cofactor activity by using in vitro sumoylation assays.