Abstract: OBJECTIVE To establish an HPLC method for the determination of vitamin B₁ tablets and active pharmaceutical ingredient related substances. METHODS Waters liquid chromatography column Symmetry C₁₈(250 mm×4.6 mm, 5 μm) was used. Mobile phase A was 4.045 g·L^-1 sodium heptane sulfonate solution (containing 1% triethylamine, pH adjusted to 3.5 with phosphoric acid), mobile phase B was methanol-acetonitrile (1:1). Gradient elution was used with a wavelength of 254 nm and a flow rate of 1.0 mL·min^-1. RESULTS The resolution of each impurity in the resolution solution was >1.5. Excipients in tablets didn't interfere with the peaks of 8 known impurities. Limit of detection of each impurity was in the range of 0.005-0.015 μg·mL^-1. It was found that the impurity H and the unknown impurity increased in different degrees under various conditions. CONCLUSION The method can be applied to the detection of related substances of vitamin B₁ active pharmaceutical ingredient and tablets. Vitamin B₁ tablets shall be stored under the conditions of cool, dry and dark.