Valosin-containing protein (VCP) is an evolutionary conserved ubiquitin-dependent ATPase that mediates the degradation of proteins through the ubiquitin-proteasome and autophagy-lysosome pathway. Despite the central role of VCP in the regulation of protein homeostasis, the identity and nature of its cellular substrates remains poorly defined. Here, we employed chemical inhibition of VCP and quantitative ubiquitin remnant profiling to reveal the substrate spectrum of VCP in human cells. We demonstrate that inhibition of VCP globally perturbs cellular ubiquitylation and increases the ubiquitylation of a distinct set of proteins compared to proteasome inhibition. We discover ~450 substrates of VCP residing in different cellular compartments, many of which are chromatin-associated, and demonstrate widespread proteasome-independent functions of VCP. Moreover, we show that VCP mediates the degradation of the transcription factor c-Myc and thereby regulates its activity. In summary, our study identifies proteasome-dependent and independent substrates of VCP, and serves as a resource for further functional investigations of VCP and its substrates in different cellular processes.