We validated and used a high-performance liquid chromatography procedure for the determination of four different amino acid neurotransmitters in cultured rat neurons and used culture medium. Samples were derivatized using 2,4-dinitrofluorobenzene and the amino acids were separated on a C18 column. The method yielded good reproducibility and sensitivity for the quantification of the four free amino acid neurotransmitters, with average recovery factors of 80.25–118.43%, an intraday precision of 0.09–0.17%, and an interday precision of 0.62–0.74%. The assay method can be readily utilized as a precise, sensitive, and highly accurate method for the determination of concentrations of the four amino acid neurotransmitters in cultured rat neurons and used culture medium. Using the described methods, we found that the aspartate, glutamic acid, glycine, and γ-aminobutyric acid concentrations (µmol/g protein) in cultured rat neurons were 25.23±0.81, 35.16±0.32, 77.56±4.51, and 62.87±3.12, respectively, whereas their concentrations (µM) in the used culture medium were 18.18±0.82, 24.27±1.01, 107.18±9.56, and 35.78±2.98, respectively.