Cancer stem cell (CSCs) are defined as a small subset of cells within a tumor with the ability to self-renew and often drive tumor progression and recurrence after chemotherapy treatment. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for patients with metastatic disease. Traditionally, cancer stem cells have been isolated from cancer cell lines and tumor biopsies and grown in undefined serum containing media using 3-dimensional cell cultures. Using these undefined culture conditions leads to experimental variability and suboptimal cell growth. 3dGRO Spheroid Medium is a new chemically defined and serum-free cancer stem cell media that supports the expansion of CSC′s in 3D spheroid cell cultures. The media supports serial passaging of cancer cell lines leading to an overall enrichment of CSC populations.