Reverse high performance liquid chromatography was used for the determination of 10-hydroxy-2-decenoic acid in royal jelly. Chromatographic separation was performed using ZORBAX Eclipse XDB-C18 column with methanol and 1% phosphoric acid (50:50, v/v) as mobile phase at a flow rate of 1.0 mL/min. The UV detection wavelength was fixed at 210 nm. This method was proved to be simple, rapid and has good reproduction quality and high recovery rate.