The intact and native top-down mass spectrometric (TDMS) techniques may be used to accurately characterize the whole sequence of intact insulin and its analogues. Diabetes, a long-term metabolic disease marked by high blood sugar, requires careful management of insulin and its analogues. Proper dosing, monitoring the efficacy of insulin therapy, and avoiding problems like hypo or hyperglycemia all depend on accurate measurement of insulin and its analogs including lispro, aspart, glargine, and glulisine. For the study of intact protein MS (mass spectrometry) has evolved into to mature and appealing technique. ESI-CID MS/MS analysis of intact insulin analog molecules can be useful in differentiating those that have very similar or identical weight and structure. Moreover, the incorporation of quadrupole - time-of-flight (TOF) analyzers greatly enhances the significance of mass spectrometry, by offering accurate ion mass measurements, enhancing resolution, and facilitating rapid analysis of complex samples. The aim of my research work was to investigate the structural analysis of human insulin and its analogues using tandem mass spectrometry (sequence analysis, differentiation between very similar sequences). I gained a substantial amount of experience with TDMS. These fragmentation studies could be useful later to identify changes in sequence or post-translational modifications that could occur during the production or storage of these pharmaceuticals.