Abstract: OBJECTIVE To establish a method to determine the content and related substances of labetalol hydrochloride. METHODS HPLC was adopted on an Agilent TC-C₁₈ column(250 mm×4.6 mm, 5 μm) using an isocratic mobile phase consisting of methanol-0.1 mol·L^-1 monosodium phosphate (55∶45) with a flow rate of 1.0 mL·min^-1. The column temperature was kept at 50 ℃ and the detection wavelength was set at 230 nm. RESULTS The resolution between the peaks of labetalol hydrochloride and labetalone hydrochloride and other peaks was good. The concentration-response relationship was linear over the range of 43.39-390.5 μg·mL-1(r=1.000). The average recovery was 99.8%(RSD=0.50%, n=9) and the limit of detection was 0.853 6 ng. The solution was stable for 24 h. CONCLUSION The method is specific, accurate, sensitive and can be used for the determination of content and related substances of labetalol hydrochloride.