Post-translational modifications, such as SUMOylation, play a crucial role in regulating various cellular processes. Traditionally, in vitro SUMOylation assays are carried out using techniques like SDS-PAGE gels and Western blotting, which are often time consuming. These informative methods, which quantitate the increase in substrate’s molecular weight, can introduce potential errors and thus a need arises for a more efficient and accurate alternative. We propose a real-time detection method for SUMOylation that utilizes a fluorescence-tagged substrate, which has been recently developed by our group (Tripathi and Das, CP Protein Sci 101:e111, 2020). We believe this method has the potential to significantly enhance the efficiency and accuracy of SUMOylation assessment, thereby advancing our understanding of SUMO signaling and its function.