For infants born with congenital anomalies, an exclusive human milk diet can reduce mortality, morbidity and improve quality of life, but their mothers are pump-dependent from birth and may experience challenges with milk production. Thus, these infants are at risk of not receiving an exclusive human milk diet and/or a long duration of human milk feeds.Antenatal milk expression (AME) is hand expression during pregnancy to collect colostrum and may support the lactation journey of these dyads. However, previous research has not clearly demonstrated adherence to AME and provided only subjective data on milk production and secretory activation. This dissertation used a prospective, longitudinal, observational design to describe AME and milk supply development in mothers of infants with congenital anomalies from 37 0/7 weeks gestation to postpartum day 14 via three aims that: 1. Examine adherence of participants willing to follow an AME recommended protocol 2. Verify the amount of colostrum that participants collected and stored through AME 3. Measure 24-hour milk supply and secretory activation onset by milk volume and human milk biomarkers of sodium and lactose A small group (antepartum, n = 19; postpartum, n = 16) of low risk women of infants with congenital anomalies were willing to initiate (94.7%) and maintain adherence (83.3%–100%) to an AME recommended protocol to attempt colostrum collection. Milk removal (80%–100%) was higher than no milk removal (0%–20%) from AME. The 24-hour colostrum volume (Median 0.35 mL, IQR 0.065-0.845) and the total colostrum volume (Median 3.99 mL, IQR 1.35-6.82) from AME varied. Following birth, four (25%) mothers experienced secretory activation onset. Pumping ≥5 times/day was manageable for 24-hour milk volume (Median 490 mL, IQR 329–6854) from days 5 to 14 postpartum, but only nine (56.25%) mothers obtained ≥500 mL of milk per day by postpartum day 14. Antenatal milk expression likely does not hasten time to secretory activation but offers an opportunity to build breastfeeding self-efficacy and duration by learning of biomechanics and skills involved for lactation.