In humans cells three SUMO paralogues (SUMO-1, SUMO-2 and SUMO-3) and six SUMO specific proteases (SENP1-SENP3 and SENP5-SENP7) are expressed. Together the SUMO proteases perform three distinct functions. They: (1) process the immature pro-SUMO proteins into the active forms, (2) remove SUMO molecules conjugated to protein targets, and (3) depolymerise SUMO conjugated within polymeric chains. By regulating these processes the SENPs play a crucial role in regulating the sumoylation state of target proteins in cells, and therefore are academically and pharmacologically interesting enzymes. Gel-based techniques for SENP analysis are well established and can be used for many applications, but their laborious methodology makes them cumbersome tools for kinetic analysis or inhibitor screening. Therefore in vitro FRET-based assays have been developed to test the three major functions of the SENPs. These use fluorescent protein fusions of the SUMOs, and together facilitate high-throughput, real-time analysis of the three major SUMO protease activities.