Whey proteins are crucial for many functions of the human body. Determining the structural properties of the protein with the enzymatic hydrolysis makes it possible to improve food quality, identify allergens and better understand food poisoning. In this study, the structural alterations of β-Lactoglobulin (model whey protein) were investigated during proteolysis. Trypsin was used as a model enzyme. Digestion of β-LG by trypsin at different concentrations were measured by Fourier transform infrared (FTIR) spectroscopy as well as by Raman spectroscopy to identify the degradation of the protein and to verify the enzymatic reactions results at various temperatures in real time. Afterwards, the advanced analysis techniques, two-dimensional correlation spectroscopy (2DCOS) and curve-fitting analysis, were applied. In addition, the experimental measurements were supplemented with DFT simulations. Based on the FTIR spectroscopy results, the most notable changes take place in the amide I (1600-1700 cm-1) and in the amide II (1480-1580 cm-1) regions. FTIR spectroscopic results revealed that the structural elements of β-LG broken down and degraded during the enzymatic digestion. Moreover, the carboxylate groups (COO-) gives rise in the infrared range (1605-1580 cm-1) as released products. Raman spectroscopic results demonstrate that β-LG loses its secondary structure and the product is formed around 1425 cm-1 arising from the carboxylate groups (COO-) due to the digestion. DFT results show that the Raman spectrum of single unit arginine and lysine residues can be predicted by DFT method. Furthermore, DFT calculations give the rise at 1683 cm-1 and 3540 cm-1 caused by C-N vibrations and N-H vibrations arising from the amino groups (NH2+), respectively.