Milk whey is considered a by-product of great importance in the industry because of its nutritional composition and its large volume of production. In this work it was studied different enzymatic associations for the serum proteolysis. In the serum prepared from pasteurized milk, by rennin addition, 6.07 mg/mL of proteins and 1.28 mmol/mL of reducing sugars were determined. Optima temperature and pH values obtained for the alcalase were 70ºC and 9.0, respectively. Km and Vmax using serum as substract were 0.66 g/L and 0.33 mmol/min/mg prot. The reaction product liberated by the proteinases was analyzed by SDS-PAGE and High Performance Liquid Chromatography (HPLC). The enzymes alcalase, trypsin, carboxypeptidase and chymotrypsin were assayed by concomitant or sequential addition at different reaction times. The results showed that the best enzymes sequence for the proteolysis was alcalase, trypsin, carboxypeptidase, chymotrypsin added at 0, 30, 60 and 90 minutes of reaction, respectively. The HPLC analysis revealed that the amount of peptides formed after sequential hydrolysis was 5 times lower than the concomitant hydrolysis (proteases added in the beginning of the reaction). This work allowed, therefore, the establishment of a methodology for these proteins hydrolysis that could be tested in reactors hereafter.