β1i is a major catalytic subunit of the immunoproteasome, an alternative form of the constitutive proteasome, and its upregulation has been demonstrated in a variety of disease states including cancer. Our lab has developed a small molecule inhibitor of β1i, dubbed UK-101. While UK-101 causes apoptosis in cancer cell lines, it was not clear whether this apoptotic effect was directly mediated by its irreversible inhibition of β1i. Since off-target effects are major roadblocks for the development of new and effective pharmaceuticals, target validation studies in this system would assist in the further progression of β1i inhibitors towards preclinical trials. Our hypothesis was that the expression and catalytic activity of β1i is important for the growth and proliferation of the PC-3 prostate cancer cell line, therefore the apoptotic effect seen upon treatment of PC-3 cells with UK-101 was due solely to its covalent inhibition of β1i. To test this hypothesis, a number of complementary approaches were used. The expression of β1i in PC-3 cells was increased by the treatment of these cells with interferon-gamma or tumor necrosis factor-alpha, natural inducers of the immunoproteasome. The expression of β1i in PC-3 cells was decreased using small interfering RNA or short hairpin RNA, in a transient or stable manner, respectively. All of these cells were then treated with UK-101. The efficacy of UK-101 decreased in the interferon-gamma treated cells but did not change in any other the other cell lines, suggesting that UK-101 was not specific for β1i. This was confirmed using a molecular probe of the proteasome and demonstrated that UK-101 bound to other proteasome catalytic subunits. Additional experiments were performed to determine the effect of β1i on the proliferation of PC-3 cells. Simply removing the β1i using small interfering RNA reduces the viability of these cells. Other studies demonstrated that a mutation of β1i which inhibited its catalytic activity reduced the viability of cells when compared to those containing the wild type protein. Overall, our data indicate that β1i is a potential therapeutic target in prostate cancer. Further medicinal chemistry efforts will be required develop UK-101 into a truly selective proteasome inhibitor.