Aldehyde dehydrogenase (ALDH) can be used as a marker to isolate, propagate, and track normal and cancerous human colon stem cells. To determine their tumorigenic potential, tissues obtained from proximal (normal counterpart) and distal (cancerous) colon of colon cancer patients are implanted into NOD-SCID mice. In parallel, ALDHhigh and ALDHlow cells are isolated via Florescence Associated Cell Sorting (FACS) after the dissociation of distal and proximal colon tissues into a single-cell suspension. Flow cytometry for ALDHhigh and ALDHlow cells is possible with the ALDEFLUOR assay. Following cell sorting, ALDH-enriched cells are tested for their tumorigenic potential in vivo as xenografts. Owing to cancer stem cell properties, ALDHhigh cells could be propagated in vivo by serial passaging of the human tissue as xenografts and in vitro as suspension cultures called sphere cultures. In this unit, all the above-mentioned methods to isolate and propagate colon cancer stem cells using ALDH as a stem cell marker are described in detail.