The effect of homogenization alone (H-) or in combination with high temperature, short time (HTST) pasteurization or UHT processing on the whey fraction of milk was investigated using highly sensitive spectroscopic techniques. In pilot plant trials, 1-L quantities of raw whole milk (3.25% fat) were homogenized in a 2-stage homogenizer at 60°C (6.9 MPa/10.3 MPa) and, along with skim milk (s-), were subjected to HTST pasteurization (72°C for 15s) or UHT processing (135°C for 2s). Other whole milk samples were processed without homogenization and used as is, or HTST only. The processed skim and whole milk samples were centrifuged to remove any residual fat and then acidified to pH 4.6 to fractionate the whey. The dialyzed whey fractions were investigated using the circular dichroism (CD), Fourier transform infrared (FTIR), and Trp intrinsic fluorescence spectroscopic techniques. Results demonstrated that homogenization combined with UHT brought about not only changes in protein composition but also a significant loss in secondary structure, particularly in the amounts of antiparallel β-sheet and α-helix, and diminished tertiary structural contact. These structural perturbations could lead to changes in the functional properties and nutritional values. On the other hand, homogenization (H-) and followed by HTST (H-HTST) treatments caused neither chemical change nor secondary structural reduction. The disruption was evident in the tertiary structural environment and the stability of the whey protein due to the homogenization of whole milk as revealed by the analyses from the near-UV CD and the intrinsic Trp fluorescence. The following order for tertiary structural stability was derived based on these studies: raw whole > HTST, H-, H-HTST > s-HTST and s-UHT > H-UHT. The methodology established in this study can be used to gain insight into the behavior of milk proteins when processed. It also provides a new empirical and comparative approach for analyzing and assessing the impact of processing schemes on the nutrition and quality of milk and dairy products without the need of extended separation and purification, which can be both time-consuming and disruptive to protein structures.