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To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fl uorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fl uorescent donor molecule is transferred to an acceptor molecule. Effi cient energy transfer requires very close proximity, and can therefore be used as a read-out for covalent and non-covalent protein interactions. The assay described here uses bacterially expressed and purifi ed YFP-SUMO-1 and CFP-RanGAP1 as model substrates that are covalently coupled in the presence of recombinant SUMO E1 and E2 enzymes and ATP. Reactions of 25 μl volume, set up in 384-wells plates, give suffi cient signal for analysis. Consequently, this assay requires very low amounts of recombinant proteins and allows measurement of time courses in high-throughput format.