Steroid hormones are biologically active factors in human milk (HM) that influence the physical and mental development of infants. Critically, maternal psychosocial stress has been associated with changes in HM steroid composition. This work aimed to characterize the steroid hormone profile of HM and pasteurized donor human milk (DHM) and assess the interplay between maternal physical and psychosocial status, the HM steroid profile, and infant outcomes.A targeted ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to quantify sixteen steroid hormones in HM samples. HM samples from mothers of term infants (N=42) and preterm infants (N=35) were collected at (i) recovery of birth weight or achievement of complete enteral nutrition, respectively, and (ii) 6 months later as well as DHM samples (N=19) from 11 donors. The physical and psychosocial status of mothers and infant neurodevelopment and temperament were assessed through structured interviews and validated questionnaires.Fourteen steroids were detected in HM/DHM samples, with cortisol, 20β-dihydrocortisol, dehydroepiandrosterone, pregnenolone, and cortisone being present in > 48% of samples. Pregnenolone, 17α-OH-progesterone, and dehydroepiandrosterone are reported for the first time in HM. Whereas milk cortisol levels were not directly related with maternal physical and psychosocial status nor with infant development, cortisone and pregnenolone correlated positively with maternal weight gain during pregnancy and were associated to maternal well-being and infant growth. The pasteurization process may have a detrimental effect on the steroid hormone levels in HM, what might influence the development of receptors.