The single cell asymmetric division assay (SCAD assay). Utilizing cancer stem cell (CSC) markers via fluorescence-activated cell sorting, putative (pCSCs) and non-pCSCs are plated as a single cell per well in a 96-well plate. Such cells are allowed to form colonies, which are cultured to generate sufficient cells to re-analyze for the presence of the original CSC markers. If a clone reconstitutes the parent phenotype, it has demonstrated the CSC-like capacity to self-renew and differentiate. If a clone fails to grow or does not reconstitute the parent phenotype, it has demonstrated a reduced differentiation potential characteristic of non-CSCs. The SCAD assay used in parallel with the xenograft tumorigenicity assay can efficiently validate CSCs and non-CSCs.