This study aims to evaluate the impact of heat treatment on the hydrolysis kinetics of cow milk proteins and on the peptide release during in vitro dynamic gastric digestion. SDS-PAGE and ELISA techniques were employed to assess the hydrolysis of proteins over time of digestion. The evolution of the peptidome generated through dynamic digestion of heated and non-heated milk was studied at different times, using MS-based techniques (ion trap and MALDI-TOF/TOF) coupled to liquid chromatography. The peptide homology value between both samples at the end of digestion (48%) confirmed the impact of heat treatment on the identity of peptides generated during digestion, despite their identical initial protein content and being the same matrix in both cases. Heat treatment produced an increased resistance to hydrolysis by pepsin in the casein fraction. However, β-lactoglobulin was found to be more susceptible to hydrolysis. Although differences on the pattern of peptide release were found between both samples, also some common traits after digestion were observed. The regions comprised between the residues 76-93 of β-casein, where several binding epitopes are included, as well as the β-casein domains 126-140 and 190-209 were found to be resistant to pepsin.