Buspirone, an anxiolytic with minimal risk of dependence or respiratory depression, lacks extensive published data on its transfer into human milk during lactation. The objective of this study was to 1) quantify the transfer of buspirone and its active metabolite 1-pyrimidinylpiperazine (1-PP) into human milk, allowing for an estimation of maternal drug exposure to the breastfed infant, and 2) report observations of the infants exposed to buspirone via breastmilk. Milk samples and health histories were collected from nine lactating mothers who donated milk samples to the InfantRisk Human Milk Biorepository while taking buspirone. The drug concentration-time profile of buspirone and 1-PP was determined using liquid chromatography-mass spectrometry. Buspirone was below the detection level of 1.5ng/mL in all milk samples with dosages ranging from 7.5 to 30mg twice daily. However, low levels of active metabolite 1-PP were observed at 7.5mg twice daily up to 30mg twice daily. The relative infant dose (RID) calculated ranged from 0.21 to 2.17%, which is below the standard 10% threshold for infant safety. There were no reports of adverse effects in the exposed infants. The levels of buspirone observed in all participants' milk samples were exceedingly low. The subsequently low relative infant dose (RID) in the range of 0.21% to 2.17% is below the 10% threshold for infant safety, suggesting that the transfer of maternal buspirone and its active metabolite (1-PP) into human milk is clinically insignificant and poses minimal risk to a breastfed infant.