Abstract: The present invention provides methods and compositions useful in the screening for agents that modulate activity of a deubiquitinating enzyme. In one embodiment, the invention relates to methods for measuring a deubiquitination activity comprising combining a deubiquitinating enzyme (DUB) with a substrate comprising a ubiquitin moiety and a fluorescently labeled compound under conditions allowing for deubiquitinating activity and measuring an altered fluorescence polarization and/or fluorescence lifetime of the released fluorescently labeled compound The invention also relates to a substrate library comprising peptides of the formula (Z)a(X)mK(X)n(Z)b useful e.g. for the determination of the substrate specificity of a deubiquitinating enzyme.