Introduction: Polyamines are essential bioactive compounds involved in cell proliferation, differentiation, and immune maturation during early life. Human milk serves as a dynamic source of polyamines, with their concentration influenced by maternal physiological and demographic factors. This study aimed to examine the effect of maternal age on concentrations of three major polyamines – putrescine, spermidine, and spermine – in human milk collected from breastfeeding mothers across a lactation period of 0–24 months. Methods: A cross-sectional study was conducted using 30 human milk samples collected from three categorised lactation stages: 0-3 months, 4-6 months, and 7-24 months. Participants were categorised into two maternal age groups: 21–29 years and 30–39 years. Polyamine concentrations were quantified using validated high-performance liquid chromatography with fluorescence detection (HPLC-FLD) following derivatisation with dansyl chloride. Results: Putrescine concentrations were significantly higher in milk samples from older mothers (30–39 years) compared with younger mothers (21–29 years) (p<0.05). In contrast, spermidine and spermine concentrations did not differ significantly between maternal age groups. Conclusion: Maternal age appeared to selectively influence putrescine levels in human milk, whereas spermidine and spermine concentrations remained relatively stable across age groups. No significant correlations were observed in this small cohort, and findings were independent of maternal dietary intake and lactation stage. Future studies with larger, longitudinal cohorts that include crucial confounding factors are needed to clarify these associations and their clinical relevance.