A HPLC method for determining the content of tetrahydrozoline hydrochloride and its related substances in eye drops was established.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, and flow rate was 1.0 mL/min.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 aver age recovery was 99.86%, and RSD was 0.23%.The method established was successfully used to determine tetrahydrozoline hydrochloride and its related substances in eye drops.