Compromised liver function has been proposed to be involved in the etiology of many diseases in early lactation dairy cows. Examining changes in individual serum triacylglycerols (TAG) may identify previously unknown metabolic pathways involved in the etiology of disease in early lactation. We used ultraperformance liquid chromatography (UPLC) in conjunction with high-resolution accurate mass spectrometry to comprehensively and quantitatively profile individual TAG in serum. The objective of this study was to examine whether individual TAG in serum may serve as disease risk indicators of mastitis, retained placenta, or both. Serum samples were collected from 161 multiparous cows 3, 2, and 1 week before calving and at calving. For this nested case-control study, serum samples of 3 groups of cows that either developed after calving retained placenta (RP; n = 8), mastitis (MA; n = 8), or remained healthy (Healthy; n = 9) were selected and serum levels of individual TAG were measured. Using PROC MIXED, overall and individual TAG levels in serum were compared between groups. The mass spectrometry-driven strategy resulted in the identification and comparative quantification of 24 individual saturated and unsaturated TAG between C48:1 and C54:4. Total TAG levels decreased until calving (P < 0.0001). Cows that subsequently developed RP had and tended to have lower TAG levels than cows that subsequently developed mastitis (P = 0.03) or remained healthy (P = 0.12). Similarly, RP cows had lower TAG with 4 double-bonds compared with Healthy cows (P = 0.04) and MA cows (P = 0.0006), which could serve as potential RP indicator. In contrast, both diseased groups had lower saturated TAG levels compared with Healthy cows (MA: P = 0.03; RP: P = 0.05). Consequently, the ratio between TAG with 4 double bonds to saturated TAG was higher in MA cows compared with Healthy (P = 0.007) and RP cows (P = 0.01), which, thus, could be a potential MA indicator. These findings suggest changes in serum TAG levels are an early indicator for the development of retained placenta and mastitis in multiparous dairy cows.