Here, we describe a method to isolate and grow normal human mammary epithelial cells (HMECs) from reduction mammoplasty specimens in vivo. The introduction of primary breast epithelial cells directly into the milk ducts of immunocompromised mice, enables their engraftment within the epithelial-rich ductal microenvironment where they maintain their epithelial identity and response to hormonal stimuli. Taking advantage of traceable in-vivo fluorescent and luciferase reporters, this approach can be readily be used as an in vivo platform for toxicity testing to study the effect of hormonal contraceptives, endocrine-disrupting chemicals and environmental toxins on the normal breast epithelium and breast cancer cells. Downstream analyses using human-specific techniques enable histological characterization and pathway analyses within a clinically relevant humanized in vivo model of the breast epithelium.