Glutaminase C Inhibitor 11 is a potent inhibitor specifically targeting Glutaminase C (GAC), demonstrating significant inhibition of GAC enzyme activity with an EC50 value of 10.64 nM. It effectively diminishes the viability of A549 non-small cell lung cancer (NSCLC) cells, presenting an IC50 of 4.025 nM. Moreover, at concentrations of 0.1 and 1 µM, it substantially impedes colony formation and reduces intracellular glutamate levels in A549 cells. Demonstrating efficacy in vivo, Glutaminase C Inhibitor 11 at a dosage of 100 mg/kg markedly curtails tumor growth in an A549 mouse xenograft model.