Sequential window acquisition of all theoretical mass spectra (SWATH) mass spectrometry is a new proteomic approach that combines the advantages of shotgun-MS (high throughput) with those of selected reaction monitoring (SRM)-MS (high reproducibility and consistency). This study aims to show for the first time the potentials of SWATH-MS for the detection and quantification of immune and metabolism related proteins in milk from dairy cows. In this study we used individual non-fractionated milk samples from 21 healthy Holstein dairy cows (SCC < 100,000 cells/mL), which were individually analyzed with a 6600 TripleTOF-MS. Data were analyzed using Skyline 4.1, interrogating a bovine milk proteome spectral library of 1,500 unique proteins. A selected subset of 34 unique proteins present in all milk samples were relatively quantified, based on validated peptide signatures (those with at least 4 ion transitions and dopt > 0.90), which were selected as optimal candidates for protein quantitation. Thus, we pooled all milk samples and the resulting sample was analyzed 10 times (10 technical replicates) to test the reproducibility of targeted peptides detection for each of the 34 proteins. From the 34 proteins, we consistently measured 27 proteins based on 2 or more peptides signals; however, 7 proteins could only be measured based on one single proteotypic peptide. Notably, both low and high abundant proteins were characterized in this study. These include CD-14, CD-9, osteopontin, apolipoproteins A-I, A-IV and E, lactadherin, mucin-1, profilin-1 and β-2-microglobulin, as well as the major milk proteins such as lactoperoxidase, xanthine dehydrogenase, lactotransferrin, β-lactoglobulin and caseins, reflecting the high resolution and sensitivity of SWATH-MS over traditional shotgun-MS methods. In conclusion, SWATH-MS is a powerful tool that allows the identification and quantitation of biomarkers and other low abundant proteins within the complex protein background of non-fractionated milk from dairy cows.