Dysfunctional mammary gland inflammatory responses around the time of parturition contribute to immunopathology associated with mastitis. Oxylipids are bioactive lipid mediators that restore tissue homeostasis following injury by orchestrating the initiation and resolution of inflammation. The objective of this study was to develop a method to detect milk oxylipid concentrations and profile changes with respect to lactation stage. Milk samples were obtained from 11 multiparous cows at the time of calving and again at approximately 80 and 220 d in milk. Analytes in milk were obtained using solid-phase column extraction and then measured using liquid chromatography with tandem mass spectrophotometry (LC/MS/MS). The amount of oxylipids in each sample were quantified relative to internal standard abundance and calibrated against standard curves. Using this newly developed extraction and analytical methodology, a total of 31 of the 63 total oxylipids species targeted for analyses could be detected in milk samples within the limits of detection. Linoleic acid (2,419 μM) was the predominant polyunsaturated fatty acid detected in milk samples regardless of lactation state followed by arachidonic (743 μM), docosahexaenoic (89.9 μM), and eicosapentaenoic (82.14 μM) acids. The linoleic acid-derived oxylipids were the most abundant found in milk with significant increases (P < 0.05) in several hydroxyl products including 9,10-dihydroxy-octadecenoic acid during late (109.6 nM) when compared with milk samples obtained in early lactation (0.01 nM). Lipoxin A4 is an arachidonic acid-derived oxylipids with known anti-inflammatory functions that was higher (P < 0.05) in milk samples obtained at the time of calving (0.01 nM) when compared with late lactation samples (0.002 nM). These are the first observations of how oxylipids profiles change with respect to lactation stage and in response to changing concentration of fatty acid substrates in milk. A better understanding of how individual milk oxylipids affect mammary gland immunity may provide new insight of how to better control dysfunctional inflammatory responses during disease.