The rapidly growing library of ubiquitin ligase structures has yielded an unprecedented view of the diversity of ubiquitin ligase substrate targeting mechanisms. However, these approaches often overlook contributions of conserved disordered domains toward substrate selection and ubiquitylation. A subset of crucial ubiquitin ligases contains large and conserved disordered domains whose function remain completely uncharacterized. HUWE1 is once such ligase that has been demonstrated to target an expanding list of structurally diverse proteins for degradation. Ligases and putative substrates with large predicted disordered domains present challenges for biochemical characterization, hindering our understanding of how disordered domains within both substrates and ligases contribute to ligase activity. To overcome these challenges, we have developed complimentary and robust proteomics-based approaches to systematically define ligase domains that are required to target substrates. These approaches allow interrogation of all potential ligase substrates within cells. By analyzing the proteomic profiles of HUWE1-disrupted cells across multiple cell lines, we globally identify a robust set of HUWE1 substrates. Rescue experiments with a collection of domain-deletion HUWE1 mutants in knockout cells reveal proteome-wide domain-substrate interactions and identify the highly conserved UBM domain and disordered domain to be crucial for substrate targeting. Proximity labeling-based interactome characterization of these mutants further demonstrates that the conserved disordered region surrounding the UBM domain is both necessary and sufficient for binding to a subset of HUWE1 substrates. Together, we establish a cell-based system to systematically characterize domain-substrate dependencies and mechanisms at the proteome level and reveal disordered domains to be important for substrate interactions.