This unit describes protocols for culturing and subsequently enriching cancer stem cells (CSCs), also referred to as tumor-initiating cells (TICs), from human established cell lines. TICs are thought to display the major cell population in the tumor with the proliferative capacity to seed tumors, implying that they( )are critical in initiating and driving tumorigenesis. The protocols show the methods for enriching and subsequently characterizing TIC populations from a series of human tumors including glioblastoma, breast, and pancreatic tumors. Protocols evaluating the morphology, phenotypic, and functional properties of TICs are described. Long-term cultures grown either as monolayers ("TIC-low") or as non-adherent tumor spheres ("TIC-high") are generated. As a result, cells from the TIC-high culture exhibit increased expression of stem cell surface markers, such as CD133, CD44, and CD24, high aldehyde dehydrogenase (ALDH) activity, and elevated expression levels of p21, in comparison to cells from the TIC-low culture. Studying TICs by using such protocols is cost effective and is considered as a suitable and simple way for studying them.