Postpartum mothers and their healthcare providers often face the challenge of limited data regarding the safety of drug therapies during lactation. Pregnancy can lead to sustained weight gain, and obesity can negatively impact both physical and psychological well-being. The introduction of GLP-1 agonists to augment weight loss has become a topic of interest for many postpartum mothers. Our study aims to investigate the transmission of semaglutide into human milk in the first steps to ensure the safety and health of both lactating mothers and their breastfed infants. Semaglutide quantification was performed using high-resolution liquid chromatography-mass spectrometry. InfantRisk Center Human Milk biorepository released milk samples from eight women collected at 0, 12 and 24 h post-semaglutide administration. Semaglutide was extracted using protein precipitation in methanol, followed by chromatographic separation. Linear calibration curves for the method ranged between 2.5–30 ng/mL, with a limit of detection of 1.7 ng/mL and a limit of quantification of 5.7 ng/mL (LLOQ). Semaglutide was not detected in any of the collected human milk samples. A worst-case scenario of the relative infant dose (RID) was calculated using the LLOQ as the drug concentration in milk when considering semaglutide’s bioavailability and long-acting dose profile. The maximum RID projected was 1.26%, far below the standard 10% safety threshold. While questions about long-term infant outcomes, the safety of maternal nutrient intake, and the nutrient content of breast milk remain, our findings suggest that semaglutide concentrations in human milk are unlikely to pose clinical concerns for breastfed infants. These results support healthcare providers in making informed decisions regarding postpartum therapeutic interventions.