Abstract The search for inhibitors of the Ubiquitin Proteasome System (UPS) is an expanding area, due to the crucial role of UPS enzymes in several diseases. The complexity of the UPS and the multiple protein‐protein interactions (PPIs) involved, either between UPS proteins themselves or between UPS components and theirs targets, offer an incredibly wide field for the development of chemical compounds for specifically modulating or inhibiting metabolic pathways. However, numerous UPS PPIs are transient/labile, due the processivity of the system (Ubiquitin [Ub] chain elongation, Ub transfer, etc.). Among the different strategies that can be used either for deciphering UPS PPI or for identifying/characterizing small compounds inhibitors, the split‐GFP approach offers several advantages notably for high throughput screening of drugs. Split‐GFP is based on the principle of protein‐fragment complementation assay (PCA). PCA allows addressing PPIs by coupling each protein of interest (POI) to fragments of a reporter protein whose reconstitution is linked to the interaction of the POI. Here, we review the evolution of the split‐GFP approach from bipartite to tripartite Split‐GFP and its recent applicability for screening chemical compounds targeting the UPS.