F(o)rster resonance energy transfer (FRET) techniques have been widely used in biological studies in vitro and in vivo and are powerful tools for elucidating protein interactions in many regulatory cascades.FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair.Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters.SUMOylation is an important post-translational protein modification with key roles in multiple biological processes.Conjugating SUMO to substrates requires an enzymatic cascade.Sentrin/SUMO-specific proteases (SENP) act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate.Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant,Kd,and protease kinetics parameters,kcat and Km,in the SUMOylation pathway.The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.