The content of lignans of Vitex negundo L. in different medicinal parts from different harvest time was determined to make full use of the antineoplastic resources of traditional medicine Vitex negundo L.RP-HPLC was used to determine the content of anti-tumor lignans in different medicinal parts, producing areas and harvest time.The chromatog. column Kromasil ODS (250 mm × 4.6 mm, 5 μm) was used as the stationary phase, and eluted with gradient elution of acetonitrile (A)-0.1% glacial acetic acid (B) (0-10 min, 15%A; 10-40 min, 28%A).The detection wavelength was 255 nm, the column temperature was 25°C and the volume flow rate was 1 mL · min-1.Lignans VBE-1 and VBE-2 had a good linear relationship under these conditions.Among different medicinal parts, the fruits of Vitex negundo L. had the highest content of anti-tumor lignans, the stems (branches) had less content while in the root and leaves the constituents were hardly detected.Content of anti-tumor lignans from Fructus Viticis Negundo in different harvest time showed that fruits collected in Nov. had the highest content of anti-tumor lignans.The detection method is simple and accurate.The method of detecting the content of VBE-1 and VBE-2 in Fructus Viticis Negundo simultaneously has been firstly established, which lays a solid foundation for making full use of Vitex negundo L. resources.