Objective To establish a HPLC method for the determination of related substances in lysine hydrochloride and zinc gluconate oral solution. Methods The HPLC separation was performed at 30 ℃ on an Inertsil Amide column (4.6 mm×250 mm,5 μm) by gradient elution with mobile phase A [phosphatesolution (2.72 g KH2PO4and 0.46 goctane sulfonate,added with 1 000 mL)-acetonitrile (22:78)] and B [phosphate solution-acetonitrile (95:5)] at the flow rate of 1.0 mL·min-1.The clumn temperature was 30 ℃.The determination wavelength was 203 nm. Results The major peaks were well separated from adjacent related substances peaks and resolution were greater than 2.0.The theoretical plate number of major peaks and related substance peaks were greater than 3 000.Excipients and destruction test products did not interfere with the determination. Conclusion This method is practicable,specific and durable for the simultaneous determination of related substances in lysine hydrochloride and zinc gluconate oral solution.