ScopeHuman milk (HM) is rich in bioactive compounds and essential nutrients. While research has focused on lipids, minerals, immune markers, microbiota, and oligosaccharides, specific metabolites are less studied. This study uses targeted metabolomics to identify and quantify metabolites in HM and explores the impact of perinatal and dietary factors on the metabolomic profile.Methods and resultsIn a cross‐sectional study of 123 healthy lactating women, HM samples were collected up to 1 month postpartum and analyzed using the Biocrates MxP Quant 500 kit. Maternal and neonatal clinical, anthropometric, and nutritional data were collected. A total of 432 metabolites were quantified and categorized into 20 groups. The metabolomic profiles formed three distinct clusters, primarily driven by triglyceride concentration differences. Docosahexaenoic acid (DHA) levels were higher in HM from mothers with vaginal delivery compared to C‐section births and differences in hexoses were found between exclusive and mixed‐feeding practices. Maternal diets rich in lipids and animal proteins were associated with elevated amino acids, sphingolipids, and glycosyl‐ceramides.ConclusionThe HM metabolome was grouped into three clusters influenced by delivery mode, lactation practices, and maternal diet. This comprehensive analysis opens new avenues to explore HM composition and offers valuable insights for future dietary interventions aimed at modulating HM.