Abstract: Objective:To establish HPLC methods for simultaneous determination of dihydromyricetin,myricitrin,aromadendrin,myricetinin in Ampelopsis grossedentatas. Methods:The chromatographic conditions were follows-Hypersil ODS 25 μm(250 mm×4.6 mm,5 μm)was choosed as the chromatographic column,the mobile phase was the mixture of methanol:acetonitri and 0.1% phpsphoric acid in water with gradient elution,the detection wavelength at 325 nm,the flow velocity of 1.0 mL,the column temperature at 25℃.Results:The linear relationship between content and peak area was obtained during the range of 7.8~7956 μg/mL for dihydromyricetin,14.2~1174.4 μg/mL for myricitrin,2.9~290 μg/mL for aromadendrin,0.7~46.5 μg/mL for myricetinin. RSD's founds of peak area of dihydromyricetin,myricitrin,aromadendrin,myricetinin for precision test,the stability test and the repeatability test were less than 2.0%. The recoveries,average recovery and RSD's of dihydromyricetin,myricitrin,aromadendrin,myricetinin obtained were 98.00%~100.12%,98.95%,0.85%;98.75%~101.86%,100.14%,1.07%;99.14%~101.24%,100.21%,0.84% and 98.25%~99.45%,98.81%,0.59%. By using the established HPLC methods,85 samples of Ampelopsis grossedentata in the same harvest period and different habitats were analyzed. The results showed that the contents of dihydromyricetin,myricitrin,aromadendrin,myricetinin were 0.3003%~34.1934%,0.0722%~1.7105%,0.0033%~1.167%,0.0007%~0.3309%. 24 samples of Ampelopsis grossedentata in the same habitat and different harvest periods were analyzed. The results showed that the contents of dihydromyricetin,myricitrin,aromadendrin,myricetinin were 0.3362%~7.6490%,0.0391%~2.9616%,0.0017~0.8361%,0.0019%~0.1050%. Conclusion:The method could be applied to simultaneous quantitative assay of dihydromyricetin,myricitrin,aromadendrin,myricetinin in Ampelopsis grossedentatas with good specifivity,reproducibility and stability. The sampling recovery test is in compliance with the requirement.