A reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV-detection at room temperature has been developed for the analysis of insulin and its main degradation product, A21-desamido insulin. Octadecylsilica was used as stationary phase and a mixture of water and acetonitrile containing tetrametylammonium hydroxide as eluent. The method produced linear response over the concentration range of 10-100 microg/ml, with an average accuracy of 97.35+/-1.36% as well as average intra- and inter-day variations of 1.29 and 5.24%, respectively. The limits of detection and quantitation of the method were 0.25 and 0.75 microg/ml, respectively. Considering the analysis specifications, the system is suitable for direct analysis of routine formulations and stability studies. By this method human insulin can be separated from its principal degradation product, A21-desamido insulin. Also there is no need for any particular requirement and it is easily available in most of laboratories.