The proteasome is a large protein complex responsible for degrading misfolded or damaged proteins tagged with ubiquitin modifications, thereby maintaining proteostasis in all eukaryotic cells. Before these proteins enter the proteasome for proteolysis, ubiquitin modifications are removed by a group of enzymes known as deubiquitinases (DUBs). DUBs can either cleave ubiquitin directly from substrate proteins or disassemble ubiquitin chains. They exhibit distinct activities and preferences for a wide variety of complex ubiquitin chains with different linkages and architectures. Understanding how proteasome-associated DUBs achieve selectivity for specific substrates is essential for elucidating the regulation of the ubiquitin system. This dissertation presents our efforts to investigate the profile of ubiquitin conjugates on the proteasome, as well as the impact of the proteasome-associated DUBs, UCH37 and USP14, on proteasome composition and stoichiometry. Our lab previously reported that UCH37 can enhance its debranching activity towards K48-linked branched ubiquitin chains when it is in the context of the proteasome. Previous studies on USP14 showed its en bloc cleavage on ubiquitin chains when proteasome is present. Despite the established roles of these two DUBs, the specificities and contributions to proteasome function remain poorly understood. Therefore, this thesis addresses how the depletion of UCH37 and USP14 affect proteasome composition and stoichiometry, as well as proteasome substrates. In our current study, we utilized CRISPR editing to generate various proteasome cell lines, purified proteasomes to characterize their activity and assembly, and then assessed ubiquitin profiles and substrates using mass spectrometry. Our results suggest that our purification method is efficient and effective to obtain fully assembled 26S proteasome. Additionally, the ubiquitin western blots imply reduced proteasome ability to process ubiquitinated conjugates. Furthermore, using DUB-based analysis and middle-down mass spectrometry, we studied ubiquitin linkage types and abundance on the proteasome. Our results indicate that proteasome substrates are mostly mono-ubiquitinated or modified with short K48-ubiquitin chains. Lastly, we identified a set of proteasome substrates and interactors are correlated with the depletion of DUBs, as well as changes in the protein abundance of proteasome subunits, using quantitative proteomics. Our results imply that DUBs act on their ubiquitinated substrates to regulate the degradation function of the proteasome. Overall, these findings advance the understanding of DUBs and proteasome substrates and will aid in developing biochemical strategies to explore their roles in various cellular processes.