Publisher Summary The chapter illustrates that the studies of hydrolytic agents have led to major advances in our knowledge of proteins, including the identification of the amino acid constituents of proteins and the development of the polypeptide concept of protein structure. These studies are now, for the most part, of historical interest, and provide an insight into protein hydrolysis. This is largely the result of three significant developments: (1) the discovery of chromatographic and electrophoretic methods for the examinatioin of the hydrolytic products of pure proteins; (2) the use of acids and enzymes as reagents for the systematic degradation of proteins into small peptides that are amenable to sequence analysis; and (3) the preparation of highly purified proteolytic enzymes that can be used for selective hydrolysis. The chapter mainly deals with the recent developments in the use of acids and enzymes for the hydrolysis of proteins, with particular emphasis on the hydrolytic methods, which are employed in amino acid sequence studies. Particular attention is given to those factors that must be considered in selection of a suitable hydrolytic reagent.