A new, rapid, and specific reversed phase high-performance liquid chromatographic (RP-HPLC) method involving precolumn derivatization with benzoyl chloride was developed and validated for the estimation of γ-aminobutyric acid (GABA) in rat brain tissue preparations. The derivatization product of GABA was identified by melting point, infrared, and proton nuclear magnetic resonance (1H NMR) spectroscopy to be n-benzoyl GABA. Various parameters which influenced derivatization and elusion were optimized. The chromatographic system consisted of C-18 column with ultraviolet (UV)—photodiode array detection ranging from 210 to 400 nm. Elution with an isocratic mobile phase consisting of 0.025 M disodium hydrogen phosphate buffer—methanol (65:35, v/v; pH 6) at a flow rate of 1 mL min-1 yielded sharp and specific peak of n-benzoyl GABA within 7 min. The method was validated with respect to the linearity, accuracy, precision, sensitivity, selectivity, and stability, wherein the benzoyl derivative of GABA showed stability for 2 months. The lower limit of detection was 0.5 nmol L-1. This novel derivatization procedure for the estimation of GABA with benzoyl chloride was also applied for rat brain tissue preparations that gave highly specific peak and good component recovery. The results show that the method for the determination of GABA by benzoylation using RP-HPLC has good linearity, accuracy, precision, sensitivity, and specificity and is simple and economical to perform.