Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors.