Background Despite the roll out of maternal antiretroviral therapy, HIV infections continue to occur in infants through breastfeeding. In high income countries, to breastfeed or not to breastfeed has been in the last decade a difficult question for women with HIV. Undetectable = untransmittable (U = U) is applicable to treatment as prevention but may not apply to breast-milk transmission.On the other hand, there is evidence that breast milk microbiota has the potential to protect against vertical transmission of HIV. Objective To analyze breast milk factors (such as HIV reservoir and microbiota) that are associated with vertical transmission of HIV to help design specific preventive strategies. Methods This is a descriptive study nested in a prospective cohort among pregnant and breastfeeding women living in an area of high HIV prevalence from southern Mozambique. Available breast milk samples from 200 women (100 living with HIV and 100 without), collected at the early post-partum period will be analyzed. One year after childbirth, an additional breastmilk sample will be collected from participants and maternal HIV viral load and infant’s HIV status assessed. Expected results The identification of both risk and breast milk protective factors of vertical transmission HIV.