Glioblastoma (GBM) is a highly infiltrating, aggressive brain cancer with no available curative treatment. We developed a rapid assay for assessing the effect of various drugs on GBM stem cells. The assay uses a small number of separated CD133+ cells (20,000 in 0.2 ml) in 96-well plate that form neurospheres within 1-2 days. Various drugs disperse the neurospheres within 24-36 h, which can be quantified microscopically. We used the GBM cell line A-172 to develop the conditions for the assay, utilizing Gleevec, the gamma-secretase inhibitor DAPT, and the anti-bacterial peptide amph1D. The results show dispersion of the neurospheres leading to cell death, at relatively low drugs concentrations (<25 microM). Drug combination showed a synergistic effect and disruption of neurospheres under lower concentrations. We applied this assay to the CD133+ cells of surgical specimens from three patients that showed similar results. This assay facilitates a rapid test of drugs on small amounts of fractionated patient's GBM stem cells.