The metabolic alterations associated with the increase in milk production make the transition period critical to the health of dairy cows, usually leading to a higher incidence of disease in periparturient animals. In this manuscript, we describe the use of NMR-based untargeted metabolomics to follow how these changes impact the serum metabolome in a group of 28 transition dairy cows with no initial clinical diseases. Principal component analysis (PCA) of serum 1H NMR data from four weeks before calving to 8 weeks after parturition allowed us to clearly identify four stages during the transition period. Pairwise comparisons using orthogonal partial least square discriminant analysis (OPLS-DA) and univariate data analysis led to the identification of 18 metabolites that varied significantly through these stages. Species such as acetate, betaine, and creatine are observed early after calving, while other markers of metabolic stress, including acetone, β-hydroxybutyrate (BHB), and choline, accumulate significantly at the height of milk production. Furthermore, marked variations in the levels of lactate, allantoin, alanine, and other amino acids reveal the activation of different gluconeogenic pathways following parturition. Concomitant with a return to homeostasis, a gradual normalization of the serum metabolome occurs 8 weeks after calving. Correlations of metabolite levels with dietary and metabolic adaptations based on animal parity could also be identified. Overall, these results show that NMR-based chemometric methods are ideally suited to monitor manifestations of metabolic diseases throughout the transition period and to assess the impact of nutritional management schemes on the metabolism of dairy cows.