Abstract: AIMTo determine insulin and its related substances in insulin powder for inhalation by reversed phase high performance liquid chromatography method. METHODSThe initial mobile phase was solution A (0.1% trifluoroacetic acid-acetonitril 70∶30) and changed to solution B (0.1% trifluoroacetic acid-acetonitril 60∶40) in 30 minutes. The flow rate was 2.0 mL·min^-1, the column temperature was 30℃, the wave length was 280 nm, the injected volume was 20 μL. RESULTSInsulin was well separated from other peaks induced in different conditions. There was good linear relationship between the amount of insulin and its peak area, the RSD was 1.1%, the insulin solution for determination was stable in 12 hours, and the quantity detected was near the added. CONCLUSIONThe method is simple and accurate to detect insulin and its related substances in insulin and its preparations.