Abstract: Objective: Setting tetrahydropalmatine as an internal reference, to establish a quantitative analysis of multi-components by single marker (QAMS) method for the determination of five alkaloids contents in vinegar Corydalis yanhusuo. Method: HPLC was employed using gradient elution with a mobile phase consisting of acetonitrile-water (containing 0.1% phosphoric acid, pH6.0). The methodological investigation was conduct to estalish a proper method. According to the relationship between the content and the chromatographic peak area of each component, calculated the relative correction factors (RCF) of protopine, palmatine hydrochloride, berberine hydrochloride and corydaline to tetrahydropalmatine. And then, the contents of five alkaloid components were determined in the various samples. Meanwhile, external standard method (ESM) was used to determine the contents. The variation in the results obtained from QAMS and RCF was statistically analyzed to confirm the precision and practicability of QAMS. Results: The linear relationship between the five alkaloids was good in a certain linear range, and the precision, stability, and repeatability met the required standards. The average recovery ranged from 98.38% to 101.28%. The RCF values of protopine, palmatine hydrochloride, berberine hydrochloride, and corydaline to tetrahydropalmatine were 0.405, 1.868, 1.571, and 1.049, respectively. There was no significant difference in the content of the five alkaloids measured by QAMS and ESM in 10 batches of processed Corydalis yanhusuo from different regions after vinegar processing. Conclusion: The developed QAMS method in this study could be effectively used for the quantitative analysis of five alkaloid components in vinegar Corydalis yanhusuo. The results were highly accurate and the method was considered feasible. This method could be served as a valuable reference for the quality evaluation of raw and processed products of TCM, containing alkaloids or multi-indicator components.