OBJECTIVE To establish a HPLC method for determining the content of tetrahydrozoline hydrochloride and its related substances in eye drops.METHODS A diamonsil C₁₈ column(4.6 mm×150 mm,5 μm)was used with a mobile phase of methanol-acetate buffer(40∶60).The detection was carried out at 235 nm.RESULTS The determination limit was 0.22 ng.The standard curve was linear in the range of 9.94-497.0 mg·L^-1(r=0.999 9).The average recovery was 99.86% and RSD was 0.23%.CONCLUSION The method established is successfully used to determine tetrahydrozoline hydrochloride and its related substances in eye drops.