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Breastfeeding is strongly recommended for its benefit to the mother and the infant. However, in HIV infection, breastfeeding can transmit the virus from the mother to the infant. The development of affordable antiretroviral medications which accumulate in breast milk may offer a novel approach to reduce the rate of vertical transmission of HIV. However, little information exists on antiretroviral pharmacokinetics in the breast milk of women, and the relationship of these concentrations to infant exposure. The focus of this work was to overcome the complexity of the milk matrix in an efficient way for sample clean-up prior to analysis. After validation of these antiretroviral extraction methods and assays, simultaneous quantification of antiretroviral in the breast milk and plasma of Malawian women were performed.