This second edition provides an overview of recent developments and approaches used by researchers to investigate the properties and functions of mammary epithelial and stem cells, which will contribute to understand the heterogeneity of the mammary gland and of breast cancer. Chapters detail processes used to characterize stem cells, single cell RNA sequencing, computational methods, sophisticated imaging techniques, and a variety of model systems, among others. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Mammary Stem Cells: Methods and Protocols, Second Edition aims to make available protocols used to navigate the intricate behavior of mammary stem cells and to gain further knowledge to take us closer to the design of innovative strategies to prevent and treat breast cancer.