Objective To develop a method for content determination of sinensetin,eupatorin,and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone in Orthosiphon stamineus. Methods The determination was carried out on a Symmetry C18 column (4.6 mm×250 mm,5 μm) by HPLC.The mobile consisted of acetonitrile and water containing 0.05% H3PO4 in gradient elution.The flow rate was 1 mL·min－1,the column temperature was 35 ℃,the detected wavelength was set at 365 nm, and the injection volume was 10 μL. Results The peak areas and the sample quantity of the three components presented good linear relationship in the range of 0.50－5.00 μg for sinensetin,0.50－5.00 μg for eupatorin,and 0.05－0.50 μg for 3′-hydroxy-5,6,7,4′-tetramethoxyflavone.The average recoveries were 101.26%,100.28% and 99.66%,respectively. RSD were 1.73%, 0.82% and 1.68%, respectively. Conclusion The method is proved to be simple,accurate and can be used for the quality evaluation of Orthosiphon stamineus.