AIM: To study the determination of 5 kinds of cholic acid content. METHODS: Using single standard substitue (SSS), we assayed 5 kinds of cholic acid, such as cholic acid, deoxycholic acid, hyodexycholic acid, chenodeoxychoilc acid and ursodeoxycholic acid by HPLC/ELSD. and studied the influence of the additive substitutes (such as rabbit Serum, urine and Qingkailing extract) on the measure values. RESULTS: By using any one of the 5 kinds of cholic acid standard, the content in all the 5 kinds of cholic acid could be determined. CONCLUSION: Simply as well as credibly, the SSS method could be used in quality analysis, especially in complex matrix. Also, it proved a feasible way in quality control and research of fingerprint chromatography of TCM.