This paper is aiming to present the validation of proposed HPLC method for simultaneous quantification of the both pinoresinol 4-O-beta-D-glucopyranoside and vladinol F in the fruits of Pandanus tonkinensis with the following conditions: using RP-C18 (250 mm × 4.6 mm; 5 μm) column, detection wavelength measured at 228 nm, the mobile phases consisting of acetonitrile and 0.1% acetic acid in gradient mode with a flow rate of 1mL / min and injection volume was 10 L Method validation showed a high specificity, linear calibration ranging from 25.5 to 76.4 g / ml (r= 0.9991) and from 26.0 to 77.9 g / ml (r= 0.9987) for two markers, respectively.