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Glucagon (a polypeptide hormone) plays a key role in treating severe hypoglycemia. It is a single chain polypeptide that contains 29 amino acid residues. Sensitive quantification assays of Glucagon in biological matrices enable us to develop a better correlation between diabetes as a function of disease progression and/or drug treatment. Typically, bioanalysis of Glucagon, like most other biologics, is performed by ligand binding assays, however, such assays can suffer from cross-reactivity issues and lack of standardization. LC-MS/MS offers some significant advantages in developing universal, robust, sensitive, and reproducible assays with shorter developmental times. In addition, LC-MS/MS can distinguish between closely related species, metabolites, or endogenous interferences.