These fluor-based substrates are superior to current stop time gel densitometry assays which are not scalable and are time and substrate consuming. However, DUB substrate specificity is influenced by many parameters and variables. These include but are not limited to: isopeptide linkages, fluor labels, tags and label/tag positions. Due to these variables, there is no universal diubiquitin substrate for all DUBs and variables must be optimized for each DUB. Each substrate in this kit varies in the position of the fluorophore and quencher and must be empirically tested for individual enzymes. Below representative data is shown indicating the optimization of the label position on various substrates for USP5 (Isopeptidase T, E-322). Labeling at the same positions with alternative quench pairs (TAMRA/QXL) further improves efficiency and signal to background. Optimized quench pairs result in a more efficient substrate, a hydrolysis rate closer to wild-type ubiquitin and increased signal to background.