An HPLC method for determination of content and related substances of Ambroxol Hydrochloride Injection was established.The Welchrom-C18 column (4.6 mm×200 mm, 5 μm) was adopted.The mobile phase was 0.01 mol·L-1 diammonium phosphate (adjusted the pH to 3.0 with phosphate) -acetonitrile (50:50) , the flow rate was 1.0 mL·min-1, and the detection wavelength was 248 nm.Ambroxol hydrochloride showed a good linear relationship within the range of 0.091-0.212 mg·ml-1 (r=0.9999), and the average recovery was 99.60% (n=9).This method was simple, accurate and reliable for the determination of Ambroxol Hydrochloride Injection and its related substances.