In the present study, the oleanolic acid (OA) and ursolic acid (UA) contents ofHedyotis diffusa and H. corymbosa were determined by a rapid, selective and accurate method combining modified ultrasound-assisted extraction (MUAE) and HPLC. Compared with traditional extraction methods, MUAE reduced the extraction time, the extraction temperature and the solvent consumption and maximized the extraction yields of OA and UA. Furthermore, the combined MUAE-HPLC method was applied to quantitate OA and UA in plant samples and exhibited good repeatability, reproducibility and stability. The mean recovery studies (one extraction cycle) for OA and UA were between 91.3 and 91.7% with RSD values less than 4.5%. The pioneer method was further applied to quantitate OA and UA in six samples of H. diffusa and five samples of H. corymbosa. The results showed that the OA and UA content in the samples from different sources were significantly different. This report is valuable for the application of H. diffusa and H. corymbosa obtained from different regions in clinical research and pharmacology.