Cancer stem cells (CSCs) are tumor cells that share functional characteristics with normal and embryonic stem cells. CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo- and radiotherapy, with important roles in tumor progression and the response to therapy. Thus, a current goal of cancer research is to eliminate CSCs, necessitating an adequate phenotypic and functional characterization of CSCs. Strategies have been developed to identify, enrich, and track CSCs, many of which distinguish CSCs by evaluating the expression of surface markers, the initiation of specific signaling pathways, and the activation of master transcription factors that control stemness in normal cells. We review and discuss the use of reporter gene systems for identifying CSCs. Reporters that are under the control of aldehyde dehydrogenase 1A1, CD133, Notch, Nanog homeobox, Sex-determining region Y-box 2, and POU class 5 homeobox can be used to identify CSCs in many tumor types, track cells in real time, and screen for drugs. Thus, reporter gene systems, in combination with in vitro and in vivo functional assays, can assess changes in the CSCs pool. We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics, demonstrating their value in CSCs research.