OBJECTIVE: To establish a method for the related substance determination of Sodium Formononetin-3′-Sulfonate. METHODS: HPLC was performed on the column of Agilent C18 with the mobile phase A of acetonitrile and mobile phase B of potassium dihydrogen phosphate solution at the flow rate of 0.8 mL/min, sample size was 10μL, detection wavelength of 250 nm and column temperature of 30℃. RESULTS: The main drug and related substances could be well separated and detected effectively; there was a good linear relationship between the mass concentration of formononetin and impurity A, B, C, D, E, F and G and peak area in the range of 0.060-4.004 μg/mL(r=0.999 8), 0.056-3.756 μg/mL(r=0.999 3), 0.039-3.902 μg/mL(r=0.999 6), 0.060-4.026 μg/mL(r=0.999 5), 0.058-3.878 μg/mL(r=0.999 3), 0.058-3.869 μg/mL(r=0.999 5), 0.060-3.977 μg/mL(r=0.999 5)and 0.040-3.952 μg/mL(r=0.999 4). respectively. The RSDs of instrument precision, intermediate precision and stability test of related substances and content were no more than 2.00%; the average recovery of impurities was in the range of 98.49%-101.76% with the RSD in the range of 0.37%-1.37%(n=9). CONCLUSIONS: The method is simple, reliable and accurate, and can be used to detect related substance in Sodium Formononetin-3′-Sulfonate.