The primary characteristics of adult stem cells are maintaining prolonged quiescence, ability to self-renew and plasticity to differentiate into multiple cell types. These properties are evolutionarily conserved from fruit fly to humans. Similar to normal tissue repair in organs, the stem cell concept is inherently impregnated in the etiology of cancer. Tumors contain a minor population of tumor-initiating cells, called "cancer stem cells". The cancer stem cells maintain some similarities in self-renewal and differentiation features of normal adult stem cells. Therefore, various methods developed originally for the analysis and characterization of adult stem cells are being extended to evaluate cancer stem cells. Relevant methods that are used generally across normal stem cells as well as cancer stem cells are summarized. Combination of two or more of these methods for validation of cancer stem cells appears to be a promising approach for the precise isolation and analysis of cancer stem cells.