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The objective of the present work is to develop a simple and sensitive method for analysis of human insulin injection by using reverse-phase high performance liquid chromatography technique. A reverse-phase high performance liquid chromatography method with UV-detection at room temperature has been developed for the analysis of insulin from formulation. Hypersil BDS C-18 was used as stationary phase, and mobile phase consisted of 60 volume of 1 mmol sodium sulphate and 0.2% triethylamine in water, pH 3.2 adjusted by phosphoric acid, and 40 volume of acetonitrile. The eluent was monitored with a UV detector set at 214 nm with a flow rate of 1 ml/min, and sample size of 20 µl were carried out at room temperature all over the study. The method produced linear response over the concentration range of 10-100 µg/ml, with a mean recovery of 97 ± 0.31% as well as average intra- and inter-day variations of 1.35 and 5.13% respectively. The limits of detection and quantitation of the method were 0.25 and 0.75 µg/ml respectively. Considering the analysis specifications, the system is suitable for direct analysis of routine formulations and stability studies. [ABSTRACT FROM AUTHOR]