Assays of the affinity of a deubiquitinating enzyme for substrate, either through binding studies or determination of the Michaelis constant, K M , can shed light on substrate selectivity and the effects of mutations on substrate interactions. The difficulty in generating sufficient quantities of ubiquitinated substrate frequently presents a barrier to these studies. We describe here an alternative approach that can be used in cases where non-hydrolyzable, chemically ubiquitinated substrate analogs can be more readily generated. The substrate analog can be utilized as a competitive inhibitor in kinetics experiments monitoring cleavage of ubiquitin-AMC (Ub-AMC) by the deubiquitinating enzyme. The resulting inhibitory constant, K i , provides a reliable approximation of the K d for ubiquitinated substrate. We show how this approach can be used to assay the affinity of the yeast SAGA DUB module for nucleosomes containing monoubiquitinated H2B.