Abstract: OBJECTIVE To optimize a method for related substances determination of vitamin B₂ tablets. METHODS The Welch Ultimate AQ-C₁₈ column(4.6 mm×250 mm, 5 μm) was used. The mobile phase A was 0.1% phosphoric acid solution and the mobile phase B was acetonitrile for gradient elution. The flow rate was 1.0 mL·min^-1. The detection wavelength was 267 nm. The column temperature was 30 ℃. RESULTS The separation of impurity and principal component peak was good. The calibration curves were linear, the impurity A in range 0.008 7-0.053 5 μg·mL^-1(r²=0.998 1), the impurity B in range 0.019 8-0.812 3 μg·mL^-1(r²=0.999 8), the purity D in range 0.007 3-0.838 7 μg·mL^-1 (r²=1.000 0) and vitamin B₂ 0.011 3- 0.427 6 μg·mL^-1(r²=0.999 4). The average recoveries of impurity A, impurity B and impurity D were 100.1%(RSD=1.6%), 100.6%(RSD=2.0%) and 101.1%(RSD=1.1%). CONCLUSION This detection method has strong specificity and high accuracy, and can be used to control related substances in vitamin B₂ tablets.