Abstract: OBJECTIVE To establish a method for simultaneous determination of ventilagolin and ventiloquinone-Ⅰ from Ventilago leiocarpa Benth and to evaluate DPPH free radical scavenging for the anti-oxidant activity. METHODS HPLC method was used to determine the content of ventilagolin and ventiloquinone-Ⅰ and methodologically inspected. Taking IC₅₀ as the evaluating criterion of scavenging capability by determining the DPPH free radical scavenging rate of ventilagolin and ventiloquinone-Ⅰ. RESULTS The contents of ventilagolin and ventiloquinone-Ⅰ were 0.56% and 0.10%. The DPPH free radical scavenging capability of IC₅₀ were 1.13, 1.63 mg·mL^-1. CONCLUSION The method has good separation and good reproducibility. The method is convenient and rapid, as well as provide an experimental to intense research of Ventilago leiocarpa Benth.