SUMO can be conjugated to many substrates using only E1 and E2 (Ubc9) in the presence of SUMO, the substrate, magnesium, and ATP. These assays contain a complex mixture of reagents, and each reactant must interact with at least one or more of the other reactants. As such, extraction of relevant kinetic parameters during substrate conjugation remains difficult under conditions of multiple turnover. To address this issue, we have utilized single turnover assays for SUMO conjugation. This is achieved by isolating Ubc9~SUMO (where ‘~’ indicates a thioester adduct) in the absence of E3 and substrate using E1, E2, SUMO and ATP. In this section we describe the methods to conduct single turnover assays using Ubc9~SUMO in conjunction with substrate titrations in the presence or absence of an E3 ligase. We will also describe methods to determine pK values during conjugation. In addition, we will describe methodologies to extract kinetic parameters from these assays.