This paper describes an original analytical methodology for a simultaneous measurement of the protein conversion rate, the mean molar weight of peptide and the degree of hydrolysis in the course of proteolysis by Size-Exclusion High-Performance Liquid Chromatography. Peak area of dead volume eluents reflects the non-converted protein. The protein conversion rate is thus determined by comparing the area at a given time to the initial area. The peptide signal allows determining the peptide molar weight distribution and degree of hydrolysis of hydrolysates. As a first step, the approach was tested on the hydrolysis of bovine serum albumin, lysozyme and rapeseed albumin by Alcalase 2.4L. Values of degree of hydrolysis were also determined by TNBS and pH-stat methods. Most of the hydrolysate obtained showed relative differences < 20% with the reference methods. The method was also adapted to fit the TNBS assay. 39 experimental validation tests were analyzed by size-exclusion chromatography, TNBS and pH stat methods. 90% of the validation data show non-significant differences between the degree of hydrolysis predicted and the degree of hydrolysis measured by TNBS method. Hence, the proposed methodology can be efficient to study the process of enzymatic proteolysis while minimizing time and quantity of sample assay required.