The cancer stem cell (CSC) theory posits that only a small population of tumor cells within the tumor has the ability to reinitiate tumor development and is responsible for tumor homeostasis and progression. Tumor initiation is a defining property of putative CSCs, which have been reported in both blood malignancies and solid tumors. In order to test whether any given human tumor cell population has CSC properties, the relatively enriched single cells have to be put into a foreign microenvironment in a recipient animal to test their tumorigenic potential. Furthermore, various in vitro assays need be performed to demonstrate that the presumed CSCs have certain biological properties normally associated with the stem cells (SCs). Herein, we present a comprehensive review of the experimental methodologies that our lab has been using in assaying putative prostate cancer (PCa) SCs in culture, xenograft tumors, and primary tumor samples.