Inhibition of the ubiquitin-proteasome pathway using bortezomib has emerged as a rational strategy for the chemotherapeutic treatment of multiple myeloma (MM). However, the competitive inhibition mechanism of bortezomib^[1] results in >97% of patients becoming resistant or intolerant to treatment in the first few years, after which survival is typically less than a year^[2]. Non-competitive proteasome inhibitors, such as imidazoline scaffolds, that regulate the proteasome via a mechanistically distinct protein:ligand interaction, and that overcome resistance and are highly effective in vivo^[3], have therefore received recent interest for overcoming the limitations of competitive inhibitors. However, little is known about the site(s) of non-competitive inhibitor binding to the proteasome, or how they exert their biological activity. Here, we have employed a strategy involving immunoprecipitation and quantitative LC-MS/MS analysis of ubiquitinylated substrates, to gain insights into the non-competitive human proteasome inhibition mechanism of imidazoline scaffolds TCH-013 and TCH-165, compared to the competitive inhibitor bortezomib. Cytosolic proteins extracted from RPMI cells incubated with 1.0 µM TCH-165, 10.0 µM TCH-013, 0.1 µM Bortezomib or 1.0 % DMSO for 1 hour were treated with ubiquitin antibody conjugated to agarose beads, then eluted with 0.1 M glycine at pH 2.5 and 4 °C for 10 min. Each protein sample was then digested using trypsin, Lys-C and Asp-N. Quantitative analysis of differential protein abundances between the different treatment groups was performed via dimethyl labelling, prior to LC-MS/MS using a Thermo Q-Exactive Plus mass spectrometer. Protein identification and quantitation was achieved using Mascot database search software. Preliminary results from this study indicate that the competitive proteasome inhibitor bortezomib presents vastly different and more abundant ubiquitinylated protein profiles compared to the non-competitive inhibitors, indicating a lack of accumulation of some proteins with non-competitive inhibition, and supporting the hypothesis that imidazoline scaffold inhibitors present a drug-induced substrate specific inhibition of proteasome mediated protein degradation.