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Insulin is a well-characterized peptide produced by recombinant DNA technology that is widely used in maintaining blood glucose level in the diabetic patients. In this study, we have used vector pET-9a to express the human insulin in E. coliBL-21 DE3. The recombinant E. colicells were cultured by fed-batch fermentation (20.0 L) process which resulted in a dry cell mass of 20 g/L. Pro-Insulin was expressed as inclusion bodies (IBs) and it was 20% of the total protein measured by densitometry. The IBs were then solubilized and refolded to get the native biological structure. The proinsulin form has been converted into active insulin by enzymatic digestion using trypsin and carboxypeptidase-B. The active insulin was purified using ion-exchange and reverse phase HPLC. The final purified insulin has achieved a purity of 98% with Des-Insulin impurity ≤ 0.5%, when analyzed in analytical HPLC as per the United States Pharmacopiea (USP) method. This optimized process of recombinant human insulin may be used as a model for insulin analogues such as glargine, aspart, lispro etc.