Simple Summary Current diagnosis of myeloproliferative neoplasms (MPNs), including polycythemia vera (PV) and essential thrombocythemia (ET), is controversial due to limitations associated with the lack of reproducibility, subjectivity and the presence of common somatic mutations in the driver genes. Metabolomics represents a powerful approach to identify altered metabolites that can differentiate between disease status at the time of diagnosis. The objective of this study was to characterize the serum metabolic profile of MPNs patients (PV and ET) and compare it with healthy controls (HC) and secondary thrombocytosis (ST) patients. The analysis revealed metabolites following similar trends between PV and ET patients, as well as unique significant differences in the serum metabolite levels of MNPs patients compared to HC and ST patients. These results could contribute to better differentiate patients with these diseases from HC and ST patients. Most common myeloproliferative neoplasms (MPNs) include polycythemia vera (PV) and essential thrombocythemia (ET). Accurate diagnosis of these disorders remains a clinical challenge due to the lack of specific clinical or molecular features in some patients enabling their discrimination. Metabolomics has been shown to be a powerful tool for the discrimination between different hematological diseases through the analysis of patients' serum metabolic profiles. In this pilot study, the potential of NMR-based metabolomics to characterize the serum metabolic profile of MPNs patients (PV, ET), as well as its comparison with the metabolic profile of healthy controls (HC) and secondary thrombocytosis (ST) patients, was assessed. The metabolic profile of PV and ET patients, compared with HC, exhibited higher levels of lysine and decreased levels of acetoacetic acid, glutamate, polyunsaturated fatty acids (PUFAs), scyllo-inositol and 3-hydroxyisobutyrate. Furthermore, ET patients, compared with HC and ST patients, were characterized by decreased levels of formate, N-acetyl signals from glycoproteins (NAC) and phenylalanine, while the serum profile of PV patients, compared with HC, showed increased concentrations of lactate, isoleucine, creatine and glucose, as well as lower levels of choline-containing metabolites. The overall analysis revealed significant metabolic alterations mainly associated with energy metabolism, the TCA cycle, along with amino acid and lipid metabolism. These results underscore the potential of metabolomics for identifying metabolic alterations in the serum of MPNs patients that could contribute to improving the clinical management of these diseases.