High throughput cell viability screening assays often capitalize on the ability of active enzymes or molecules within viable cells to catalyze a quantifiable chemical reaction. The tetrazolium reduction (MTT) assay relies on oxidoreductases to reduce tetrazolium into purple formazan crystals that are solubilized so absorbance reflects viability, while other assays use cellular ATP to catalyze a luminescence-emitting reaction. It is therefore important to know how accurately these assays report cellular responses, as cytotoxic anti-cancer agents promote cell death via a variety of signaling pathways, some of which may alter how these assays work. In this study, we compared the magnitude of cytotoxicity to different cell types provoked by currently used anti-cancer agents, using three different cell viability assays. We found the three assays were consistent in reporting the viability of cells treated with chemotherapy drugs or the BH3 mimetic navitoclax, but the MTT assay underreported the killing capacity of proteasome inhibitors. Additionally, the MTT assay failed to confirm the induction of caspase-mediated cell death by bortezomib at physiologically relevant concentrations, thereby mischaracterizing the mode of cell death. While the cell viability assays used allow for the rapid identification of novel cytotoxic compounds, our study emphasizes the importance for these screening assays to be complemented with a direct measure of cell death or another independent measure of cell viability. We caution researchers against using MTT assays for monitoring cytotoxicity induced by proteasome inhibitors.