Determination of peptide fragments of proteins upon proteolysis process is crucial in many fields, including biotechnology, proteomics and food industry. Herein, our aim was to compare the structural changes of intact protein during enzymatic hydrolysis at various temperatures by using biophysical approaches. Proteolysis of β-Lactoglobulin (β-LG) was carried out with trypsin, and the changes were followed in situduring proteolysis at 37°C and 45°C by using Fourier transform infrared (FTIR) spectroscopy. Dynamical response of β-LG to enzymatic attack was identified on the molecular level. The IR signals of protein secondary structures decreased while carboxylate group signals arising from liberated products of proteolytic reaction increased in the 1605-1580 cm-1range to the different extent. FTIR data revealed that the degree of protein changes and liberated products varies greatly based on the reaction temperature. This study demonstrates the potentiality of FTIR spectroscopy for in situtracking of protein hydrolysis at various conditions.