OBJECTIVE: To establish a method for the content determination of calycosin glycoside in TCM preparation containing Astragalus membranaceus. METHODS: Using TCM preparation Yanggan yishui granules as model drug in our hospital, referring to HPLC method for content determination of calycosin glycoside stated in terms of “A. membranaceus” of 2015 edition of Chinese Pharmacopoeia (part Ⅰ) (called “pharmacopoeia” for short), use pharmacopoeia chromatogram condition and on this foundation to optimize and improve which by the method of increasing the formic acid proportion of mobile phase and prolonging the gradient elution time of the high polar segment of the mobile phase; the method was established for content determination of calycosin glycoside in TCM preparation containing A. membranaceus. Preliminary verification was carried out by using other two kinds of TCM preparation containing A. membranaceus. RESULTS: Under the chromatographic condition of pharmacopoeia, chromatographic peaks of calycosin glycoside in model drug could not be separated from the baseline. Optimized chromatographic condition included that mobile phase was acetonitrile-0.2% formic acid solution (14 ∶ 86, V/V); the detection wavelength was 260 nm; the column temperature was set at 30 ℃; the flow rate was 1.0 mL/min. Under optimal conditions, the linear range of calycosin glycoside was 5.08-81.28 μg/mL. RSDs of precision, stability and reproducibility tests were all lower than 2.0% (n=6). Average recovery was 100.22%(RSD=0.95%,n=6). Average content of calycosin glycoside in 5 batches of model drugs by this method was 0.014 2%. After validation, the method could effectively isolate calycosin glycoside from other 2 kinds of TCM preparations containing A. membranaceus. CONCLUSIONS: Established HPLC method can be used for content determination of calycosin glycoside in TCM preparations containing A. membranaceus. The method is accurate, reliable, reproducible and suitable, which lays the foundation for further research.