Glioblastoma is one of the most common malignant tumours in adults, with an average life expectancy of less than 1 year. The high mortality of glioblastomas is attributed to its resistance to conventional chemotherapeutic agents. Numerous studies have established the presence of a cancer stem population within glioblastomas. These CSC (cancer stem cell) populations express the cell-surface marker, CD133, and are tumorigenic and chemoresistant. Hence, CSCs make a potential target for anticancer therapies. We have focused on techniques that can reliably identify and isolate a viable CSC population, and studied their chemoresistant attributes. We show the presence of a CSC population within a slowly proliferating glioblastoma cell line, U138MG. An improvised neurosphere enrichment culture technique was developed for the isolation of CSC population. Stem cell neurospheres obtained by this protocol maintained their viability for several weeks, and could be redispersed for deriving colony-forming units and secondary spheres from single-cell suspensions. RT-PCR (reverse transcription–PCR), cell surface localization by immunofluorescence and enumeration by FACS analysis showed that the sphere cultures of U138MG grown on agarose-coated plates had elevated CD133 levels. Drug sensitivity assays indicated that these enriched spheres were more resistant to drug treatment than their non-CSC controls. Drug-resistant CSC had an increased expression of ABC (ATP-binding-cassette) drug transporters, such as ABCC2, ABCC4, ABCG2 and p-glycoprotein, indicative of their role in the resistance mechanisms. These studies will facilitate the development of in vitro assays for the sparse CSC population and strategies for improved treatment regimens for glioblastomas.