HPLC technique was developed for the simultaneous analysis of the major bioactive components: 3-isomangostin (1), gamma-mangostin (2), beta-mangostin (3), and alpha-mangostin (4) in T. pagdeni propolis extract from mangosteen orchard. The analysis method has been optimized from previous reports ( Walker, 2007 and Yodhnu et al., 2009) with slight modifications. From several trials, the mobile phase gradient system of 0.2% formic acid in methanol was the optimal condition. It provided symmetrical peaks and has the most efficient separation and speed. The maximum absorbance of alpha-mangostin (4) 245 nm was used for wavelength detection. The chromatograms of propolis extract and authentic compounds are shown in Fig. 1.