Bovine mastitis, predominantly caused by pathogenic Staphylococcus aureus, is a major impediment to milk production. Although its prevalence varies from cows to buffaloes, it lacks relevant markers that mark the phases and progression of the disease. Thus, the focus of this study was to identify key proteins that mark the transition from subclinical to clinical states in a species specific manner. Whey proteins isolated from healthy and S. aureus infected milk from both Holstein Friesian cow and Murrah buffalo were characterized by label free quantification using mass spectrometry. Collectively, 662 proteins were identified with atleast two peptides, of which 51 and 80 proteins were found significant by ANOVA (p value < 0.05) with increasing or decreasing trends in the disease progression in HF and Mu, respectively. Linear change in expression of proteins like haptoglobin and fibronectin in HF and spermadhesin and osteopontin in Mu correlated with the stage of the disease and progression in a species specific manner. Similarly, high mobility group proteins, angiogenin, cofilin-1 that were previously reported in oxidative stress were overexpressed whilemembers of ubiquitin family which remove aggregated proteins were downregulated during the progression of infection. Subsequently, selected differentially expressed proteins (e.g. osteopontin, CAP-1 and fibrinogen α) were validated by Western blot analysis. Results of this study provide deeper insights on whey proteome dynamics and a signature pattern indicative of disease progression.