Abstract: The objective this study was to evaluate the preparation of protein hydrolysates with an appropriate peptide profile as well as the reduction of costs for scaling-up the process, the actions of subtilisin and pancreatin on Whey Protein Concentrate (WPC) were tested. The effect of Enzyme: Substrate ratio (E:S) and Substrate Concentration (SC) was also verified. The hydrolysates were fractionated by size-exclusion-HPLC and the rapid correct fraction area method was used for quantifying peptides and free amino acids. The best peptide profiles were very similar giving rise to 13.17% of di-tripeptides, 46.41% of free amino acids and 12.31% of large peptides on an average. The advantage of using a higher SC was observed only in one case and for the pancretin action. The use of a smaller E:S ratio for subtilisin action was advantageous only when comparing 2:100 with 4:100 while for pancretin this beneficial result was also achieved when comparing either 1:100 with 2:100 or 2:100 with 4:100. It can be inferred that the use of these two enzymes was able to produce some WPC hydrolysates with appropriate peptide profiles for dietary purposes.