RATIONALE: Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetyl-aspartyl-glutamate (NAAG) to yield glutamate (Glu) and N-acetyl-aspartate (NAA). Inhibition of GCPII has been shown to remediate the neurotoxicity of excess glutamate in a variety of cell and animal disease models. A robust high-throughput LC/MS/MS method was needed to quantify GCPII enzymatic activity in a biochemical high-throughput screening assay. METHODS: A dual-stream LC/MS/MS method was developed. Two parallel eluent streams ran identical HILIC gradient methods on BEH-Amide (2x30mm) columns. Each LC Channel was run independently, the cycle time was 2 min per channel. Overall throughput was 1-minute per sample for the dual-channel integrated system. Multiply injected acquisition files were split during data review, batch metadata was automatically paired with raw data during the review process. RESULTS: Two LC sorbents, BEH-Amide and Penta- HILIC, were tested to separate the NAAG cleavage product Glu from isobaric interference and ion suppressants in the bioassay matrix. Early elution of NAAG and NAA on BEH-Amide allowed interfering species to be diverted to waste. The limit of quantification was 0.1 picomoles for Glu. The Z-factor of this assay averaged 0.85. Over 36,000 compounds were screened using this method. CONCLUSIONS: A fast gradient dual-stream LC/MS/MS method for Glu quantification in GCPII biochemical screening assay samples was developed and validated. HILIC separation chemistry offers robust performance and unique selectivity for targeted positive mode quantification of Glu, NAA and NAAG.