Ubiquitin-mediated proteolysis regulates all aspects of cellular function, and defects in this process are associated with human diseases. The limited number of identified ubiquitin ligase–substrate pairs is a major bottleneck in the ubiquitin field. We established and applied genetic technologies that combine global protein stability (GPS) profiling and genetic perturbation of E3 activity to screen for substrates of the Skp1–cullin–F-box (SCF) ubiquitin ligase in mammalian cells. Among the >350 potential substrates identified, we found most known SCF targets and many previously unknown substrates involved in cell cycle, apoptosis, and signaling pathways. Exploring cell cycle–stage stability, we found that several substrates used the SCF and other E3s in different cell cycle stages. Our results demonstrate the potential of these technologies as general platforms for the global discovery of E3-substrate regulatory networks.