Cells within the tumor are highly heterogeneous. Only a small portion of the cells within the tumor is capable to generate a new tumor. These cells are called cancer stem cells. Theoretically, cancer stem cells are originally from normal stem cells or early progenitor cells which accumulate the random mutations and undergo an altered version of the normal differentiation process. The cancer stem cell drives tumor progression and its recurrence. Thus, the technique to identify and purify the cancer stem cell is the key in any cancer stem cell research. In this protocol, we provide the basic technology of identification and purification of breast cancer stem cells as well as further functional assays to help the researchers achieve their research goals.