Abstract Most cancer cells undergo a number of metabolic changes, which dramatically alter their nutritional and energetic pathways. One such alteration is an addiction to glutamine, an abundant nutrient in blood that is much less utilized by healthy tissues. The glutaminase enzymes help cancer cells satisfy their metabolic requirements by acting as the first step in glutamine’s supply of carbon into the citric acid cycle. Two major isozymes of glutaminase exist, a kidney isozyme and a liver isozyme, and these are differentially expressed across different cancers. While the importance of these enzymes has been increasingly appreciated in the literature, and the inhibition of either enzyme has been shown to be detrimental to the growth of numerous cancers both in cell culture and in vivo, medicinal chemistry efforts to target either enzyme have largely involved just one class of compound, based around the scaffold of BPTES, which potently inhibits only the kidney glutaminase isozyme. It is generally accepted that most drugs have at least one off-target or biologic activity that was not intended. In order to determine new inhibitors of either isozyme of glutaminase, we examined the approved oncology drug set from the NIH, which included 119 FDA-approved, clinically relevant compounds. By assaying inhibition of the function of recombinantly expressed purified enzymes, and examining the production of ammonia (a byproduct of glutaminase catalytic activity) in drug-treated cancer cells, we found that several of the drugs in the library, such as cisplatin, not only inhibit glutaminase at biologically meaningful concentrations, but inhibit one isozyme much more potently than the other. This study both shows the power of screening traditional chemotherapeutic agents against more recently identified therapeutic targets and reveals several new scaffolds that might be optimized to potently inhibit these critical enzymes. Citation Format: William P. Katt, Richard A. Cerione. Traditional chemotherapeutic agents as selective inhibitors of glutaminase isozymes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A156.