Undifferentiated cancer stem cells (CSCs) with their unique potential of self-renewal and multi-lineage differentiation fuel tumour growth and relapse. Efficacy of glioma therapy can be considerably improved if the target is focused towards successful identification and elimination of CSCs. The aim of this research lies in defining specific and selective marker(s) to isolate glioma stem cells, to explore the differentiation state of brain tumour cells and to determine the protein profile changes that assist tumour cells to sustain stem cell-like characteristics. The three stem cell-related protein (CD133, Oct4-A and BMP3) expressions were investigated in control, hypoxic and serum-deprived U87-MG cells in order to shed light on the influence the micro-environment has in generating stem cells. Hypoxia offered a rapid state of undifferentiation as compared to serum deprivation by expressing a basal level of CD133 protein, a designated stem cell marker. Subsequent measurements of chemosensitivity and cell cycle analysis under undifferentiation conditions added to the cytotoxic potential of Taxol and showed an enhanced sensitivity of serum-deprived cells towards chemotherapeutic drugs. Moreover, proteomic analysis produced a wide dataset, depicting the changes that occur at the proteomic level in the differentiated and undifferentiated U87-MG cells. With ingenuity pathway analysis (IPA), human protein research database (HPRD) and the literature review, several proteins were proposed to be tested as potential biomarkers. They included Uracil DNA glycosylase (UDG), Phosphoglycerate kinase 1 (PGK1), Heterogeneous nuclear riboprotein K (HNRNPK) and moesin that can be used as differentiated markers for glioma cells. Vimentin, Eukaryotic translation initiation factor 4e (EIF4e), Casein kinase II alpha 1 (CSNK2A1) should be further investigated to study their precise role in gliomagenesis. Laminin binding protein associated with Integrin α6β1 and BMP2 should also be explored as a potential biomarker for isolation of glioma stem cells. This novel study envelops diverse aspects related to CSCs such as biomarkers, stem cell niche, chemoresistance, cell cycle and proteomics and also suggests the existence of two sub-types of CSCs within glioma population. It can be concluded that the finding thus obtained may be a step in the right direction in helping treat brain tumours.