SUMOylation (Small Ubiquitin-like Modifiers, SUMO group) is known to regulate protein–protein interactions, subcellular localization, and the activities of SUMO-modified targets. Over the past two decades, the SUMO pathway has been implicated in numerous developmental processes in Drosophila, including embryogenesis, wing morphogenesis, innate immune system development, neurogenesis, and apoptosis. Notably, the Hedgehog (Hh) family of secreted proteins activates the Ser/Thr kinase Fused (Fu) by promoting its SUMOylation, which is crucial for Drosophila development. Here, we describe cell-based assays to examine Fu SUMOylation in Drosophila cell cultures, offering a powerful tool to explore the role of SUMOylation in regulating signal transduction.