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In this study, Liquid Chromatography–Mass Spectrometry (LC-MS)-based metabolomics profiling was conducted to elucidate the urinary profiles of premature infants during early and late postnatal stages. As a result, we discovered significant excretion of maternal drugs in early−stage infants and identified crucial metabolites like hormones and amino acids. These findings shed light on the maternal impact on neonatal metabolism and underscore the beneficial effects of breastfeeding on the metabolism of essential amino acids in infants. This research not only enhances our understanding of maternal–infant nutritional interactions and their long−term implications for preterm infants but also offers critical insights into the biochemical characteristics and physiological mechanisms of preterm infants, laying a groundwork for future clinical studies focused on neonatal development and health.