Mastitis (SCM) is one of the most important infectious diseases of dairy cows associated with a considerable loss with regard to milk yield and the culling of cows. Early identification of susceptible cows can enable developing better preventive measurements ahead of disease occurrence. Currently, SCM is diagnosed through measurement of somatic cell (immune cells) count (SCC) after calving, and there are no screening tests available. Therefore, this study’s objective was to identify metabolic alterations in the serum of pre-SCM cows during the dry-off period, at -8 and -4 wks before calving, through a flow injection liquid chromatography coupled with tandem mass spectrometry (FIA/LC-MS/MS). A total of 145 multiparous dairy cows were included in this nested case-control study at the drying off and were sampled and monitored for periparturient disease occurrence at both prepartum and postpartum. Only 15 cows resulted free of disease and served as controls (CON), and 10 cows were affected only by SCM. Cows affected by other diseases or SCM and another disease were excluded from further evaluation. Results showed multiple metabolite alterations in SCM cows’ serum, including several amino acids, phosphatidylcholines, acylcarnitines, and organic acids. Metabotyping of cows revealed a total of 59 and 48 metabolites that differentiate CON and SCM cows at -8 and -4 wks prior to the expected date of parturition, respectively. Regression analysis indicated that a panel of 4 serum metabolites including alanine, leucine, betaine, and ornithine (AUC = 0.92; P < 0.001) at -8 wks and alanine, pyruvic acid, methylmalonic acid, and lactic acid (AUC = 0.92, P < 0.01) at -4 wks before parturition might serve as screening biomarkers for SCM. More research is needed to validate the two panels of metabolites identified and the development of potential pen-side tests.