Abstract: The presently disclosed subject matter relates to a method for characterizing a protein. The method can comprise disposing a protein in a digestion buffer; disposing a hydrolyzing agent inhibitor in the digestion buffer; passing the digestion buffer comprising said protein and said hydrolyzing agent inhibitor through a reaction chamber comprising at least one hydrolyzing agent, wherein said protein contacts said hydrolyzing agent in the presence of said inhibitor and is present in the chamber for a period of time (t) sufficient to produce protein fragments and provide digestion of said protein in the chamber, wherein the passing of the digestion buffer comprising the protein and the hydrolyzing agent inhibitor through the chamber is done at an adjustable flow rate; and performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) to characterize the protein.