Ubiquitylation is one of the most abundant protein modifications in cellular signaling, controlling numerous cellular pathways such as transcription, translation, vesicle transport and apoptosis. Ubiquitinlabels substrate proteins via a highly ordered multi-step enzymatic cascade, with specific differences in the length and topology of poly-Ub chains signalling a range of signalling outcomes or proteolytic degradation via the proteasome. Identification of E3 ubiquitin ligase substrates is key to defining their biological function and understanding their roles in disease. However, even with advances in proteomics and in vitro assays, substrate identification remains a significant challenge. An integrated approach was developed to define the E3 ligase substrates, combining orthogonal proteomics approaches to identify interacting proteins and ubiquitylation targets, combined with BiFC to validate substrates in situ. This approach was applied to identify substrates of the UBR5 ligase, which has been implicated in cancer progression and chemoresistance. UBR5-interacting proteins were isolated using GFP-Trap affinity purification followed by nanoLCMS/MS identification and label-free quantitation. This approach identified ~ 450 UBR5-interacting proteins, many dependent on the UBR and HECT functional domains for binding. Analysis of differentially ubiquitylated proteins in breast cancer cells depleted of UBR5 by shRNA identified ~1500 proteins with altered ubiquitylation when UBR5 is depleted. Intriguingly, a large number ofubiquitin-proteasome system components have altered ubiquitylation in UBR5-depleted cells, suggesting co-ordinate regulation of this system. Integration of the UBR5-interactome and ubiquitome generated a set of 171 high-confidence ligase substrates. A number of these were validated using BiFC and the role of UBR5 functional domains in mediating these interactions using disease-specific mutants was mapped. Importantly, a novel functional domain in UBR5 was identified and was found to be important for binding to some UBR5-interacting proteins. These orthogonal but complementary approaches provided interesting new insights into the function of UBR5, suggesting a role in mediating crosstalk between DNA damage response and transcriptional regulation, and the regulation of H2B post-translational modification cross talk.