Technical Abstract: Knowledge of milk protein composition/expression in healthy cows and cows with mastitis will provide information important for the dairy food industry, mammary biology and immune function in the mammary gland. To facilitate maximum protein discovery, milk was fractioned into whey, milk fat globule membranes (MFGM) and milk exosomes from healthy and Staphylococcus aureus infected cows. Amine-reactive isobaric tags (iTRAQ) were used to quantify protein changes between milk fractions isolated from healthy and S. aureus infected cows. Samples were subjected to offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to a tandem mass spectrometer. 2971 milk proteins combining the data from exosomes (2350 proteins), MFGM (1012 proteins) and whey (748 proteins) were identified with a false discovery rate of 0.1 %. 1490, 302 and 334 proteins were found to be unique to milk exosomes, MFGM and whey, respectively, under the conditions used. Greater than 300 milk proteins associated with host defense were identified and 94 were significantly differentially regulated in S aureus infected milk compared to their uninfected controls. These differentially regulated host defense proteins were selectively segregated in the 3 milk compartments examined. An example of this segregation of host defense proteins was the partitioning and high concentration of proteins indicative of neutrophil extracellular traps (NETs) formation in the MFGM preparations from S aureus infected milk as compared to exosomes or whey. Protein composition changes found in milk exosomes, MFGM and whey during an infection provides new and comprehensive information on milk protein composition in general as well as changes occurring during an infection. This study provides new insights into mammary immune function and milk protein composition in changing conditions associated with infection.