Abstract: OBJECTIVE Establish the method of measuring the amino acid neurotransmitters contents in rat brain tissue. METHODS Sodium acetate-acetonitrile-water was used as the mobile phase. The reversed phase HPLC system with pre-column derivatization by Phenyl isothiocyanate(PITC) and with UV detection was used to measure the contents of nine kinds of amino acid neurotransmitters including glutamate(Glu), aspartate(Asp), glycine(Gly), taurine(Tau), γ-aminobutyric acid(GABA), glutamine(Gln), tyrosine(Tyr), tryptophan(Trp) and serine(Ser) in the cerebral cortex, hippocampus, striatum, mesencephalon, cerebellum and hypothalamus of rats. RESULTS The nine kinds of amino acids were well separated using elution within 50 minutes. Good linear response was obtained for the nine kinds of amino acids within the range of 5 to 400 μmol·L^-1 with correlation coefficients not less than 0.99. The average recovery rate of these amino acids was not less than 80%. CONCLUSION The method is simple, accurate, high sensible and better repeatability. It was saitable for measuring the amino acid neurotransmitters in brain tissue in common laboratory.