Abstract: AIM To develop a reverse phase argentation high performance liquid chromatographic (RP-AHPLC) method for the separation and determination of ginkgolic acids. METHODS Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was applied to identify ginkgolic acids from Ginkgo biloba leaves and four ginkgolic acids of the samples were separated and quantified by RP-AHPLC. Leaves were extracted with ethanol and analytes were extracted with hexane after addition of acid/salt solution and adsorbent to matrix solution. Ginkgolic acids were separated and determined within 30 minutes by RP AHPLC under optimum chromatographic conditions. Methanol and 5% aqueous acetic acid (90∶10) containing 0.03 mol·L^-1 silver ion was used as mobile phase, column temperature was selected at 30℃, flow rate was 1.0 mL·min^-1 , UV detection wavelength was at 310 nm. The spectra analysis and purity identification of chromatographic peaks of ginkgolic acids were further confirmed by means of diode array detection. RESULTS Four ginkgolic acids were baseline separated from each other and from other interfering components. The average recovery and relative standard deviation of the method were 97.3% and 1.6%, respectively. CONCLUSION RP-AHPLC was an excellent method for separation of homologous with different carbon atom numbers and double bond. The method is useful for the quality control of extract of Ginkgo biloba leaves.