To establish a method using HPLC for the simultaneous content determination of geniposide and deacetyl asperulosidic methyl ester in Fructus Gardeniae. Methods:The HPLC method was applied with Agilent Eclipse XDB-C₁₈ column, The mobile phases consisted of acetonitrile and 0.1% phosphoric acid aqueous solution, gradient elution, the detection wave length was at 238 nm and the flow rate was 1ml/min. Results: Deacetyl asperulosidic methyl ester and geniposide showed a good linear relationship in the range of 0.009-0.186mg/ml(r=0.9994),0.042-0.846mg/ml(r=0.9996) respectively, the average recovery were 97.08% and 100.45% with RSD at 1.48% and1.29% (n=6). Conclusion: The method is simple, accurate and easy to operate, has good precision, repeatability and recovery. It can be used to determine contents of these two ingredients in Fructus Gardeniae.