Our understanding of the biology of stem cells and their therapeutic potential relies heavily on robust functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian central nervous system (CNS), neural stem cells (NSC) are often isolated and studied using a culture system referred to as the neurosphere assay. Since its introduction in 1992, the neurosphere system has been used to isolate, expand, and identify the presence of an NSC population, but also to enumerate NSC frequency. Furthermore, the neurosphere system has been used to investigate the effects of exogenous signaling molecules, genetic alterations, and as an assay to evaluate cell purification strategies. Recently, we challenged the central premise of the neurosphere assay – that all neurospheres are derived from an NSC – is not true, thereby precluding the use of the neurosphere assay to accurately measure NSC numbers. These results implied that while the neurosphere culture system provides a simple means to isolate and expand NSC harvested from the embryonic and adult mammalian CNS, its application as a quantitative in vitro assay for measuring NSC frequency is limited. In order to address the need for an assay that can reliably detect alterations in NSC frequency, we developed a new single-step semisolid-based assay, the neural colony-forming cell (NCFC) assay, which allows discrimination between NSC and progenitors by the size of colonies they produce (i.e., their proliferative potential). We anticipate the NCFC assay will provide additional clarity in discerning the regulation of NSC, thereby facilitating further advances in the promising application of NSC for therapeutic use.