Abstract: Objective:The aim was to establish a high performance liquid chromatography(HPLC)method with ultrasonic extraction to determine the content of betulinic acid,oleanolic acid and ursolic acid in Lycopus lucidus Turcz. var. hirtus Regel from different places simultaneously. Methods:On the basis of single factor experiment,the volume fraction of ethanol,ultrasonic power and time were selected as experimental factors,and triterpenic acid content was taken as response value to optimize extraction conditions. The chromatographic conditions were optimized by screening column types,pH of mobile phase,column temperature and flow rate. The applicability of the method was examined by the methodology. Results:The tested triterpenic acid showed a good linearity with their respective peak areasin the range of 2.558~51.150,2.480~49.590,2.480~49.590 μg/mL,r≥0.9999,the limit of determination was 5~7 ng,and the limit of quantification was 16~22 ng. The precision,reproducibility and stability were good,the average recovery was in the range of 100.52%~103.77%,and the RSD ranged from 0.99% to 2.42%. The HPLC chromatograms of Lycopus lucidus Turcz. var. hirtus Regel from different regions were similar. The content of betulinic acid,oleanolic acid and ursolic acid was different,and the species of triterpenic acid in different habitats was similar. Conclusion:This method is simple,sensitive,accurate,practical and reliable. It can be used for quantitative analysis and quality control of triterpenic acid in Lycopus lucidus Turcz. var. hirtus Regel samples. It provides experimental data for improving the current quality standard,development and utilization of Lycopus lucidus Turcz. var. Hirtus Regel.