Recent studies suggest that reducing synaptic glutamate release may prove therapeutic in schizophrenia. Phosphate-activated glutaminase (PAG), present in human brain mitochondria, is thought to medwiate the major flux of glutamate to the neurotransmitter pool. PAG catalyzes the conversion of L-glutamine (Gln) to L-glutamic acid (Glu), so inhibiting PAG seems a promising means of reducing Glu release. The goal of this study was to establish the parameters for a high-throughput screening to identify potential PAG inhibitors. Glutaminase A from E. coli was used as a substitute for PAG. Gln was added as the substrate and its conversion to Glu measured using Amplex Red technology as Resorufin fluorescence. First, the linear range for the relationship between glutamate added and Resorufin fluorescence was determined. We measured the kinetics of PAG action, showing that after one hour there was a complete conversion of glutamine to glutamate. However, the relationship between PAG concentration and Resorufin fluorescence was non-linear, consistent with cooperativity in PAG function. We then assayed 6-diazo-5-oxo-L-norleucine (DON), the best known glutaminase inhibitor, and determined an inhibition constant of 73 nM. DON was more effective against bacterial glutaminase than the known millimolar-level inhibition in mammalian neurons, indicating that the bacterial glutaminase would not be suitable for screening for inhibiters of human PAG, and thus for identifying PAG inhibitors with therapeutic potential for schizophrenia. In conclusion, the parameters for the assay of PAG activity and its inhibition were established; and synthesis of human glutaminase should allow us to move forward to high-throughput screening.