Förster resonance energy transfer (FRET) is a powerful tool in biological 
research and has been widely used in the study of biomolecular interactions. 
SUMOylation is an important post-translational modification that is involved in 
many key biological processes. As a multi-step cascade reaction, SUMOylation 
involves multiple enzymes and protein-protein interactions. Here, we report the 
development of an in vitro FRET-based high-throughput screening (HTS) assay in 
SUMOylation. This assay is based on steady state and high efficiency of the 
fluorescent energy transfer between CyPet and YPet fused to SUMO1 and Ubc9, 
respectively. We optimized the assay and performed a small-scale pilot study to 
validate the screening platform. Carried out in 384-well plate format, our 
FRET-based HTS provides a powerful tool for large-scale and high-throughput 
applications.