Gambogic acid obtained from plant resin of Garcinia species has anticancer properties. This study focuses on the development and validation of the high-performance liquid chromatography (HPLC) method for the quantification of gambogic acid (GA) content in the gamboge raw sample. The chromatographic system was performed on C18 column (150 × 4.6 mm, 5 µm). The system consisted of acetonitrile and 0.1% orthophosphoric acid (85:15 v/v) as mobile phase with a flow rate of 1.5 mL/min. The detection was carried out at a wavelength of 360 nm and the retention time of GA was evident at 9 min. The method was validated according to ICH guidelines. The obtained calibration plot exhibited good linearity in the range of 5 to120 µg/mL (r2 > 0.999). The percentage recovery GA spiked in the gamboge sample ranged from 98.87% to 102.92%. The LOD and LOQ were shown to be 2.069 and 6.271 μg/mL, respectively. The validation study demonstrated that this method was simple, specific, and applicable for quantitative analysis of GA in gamboge resin.