The efficacy of glioma therapy can be considerably improved if it eliminates cancer stem cells (CSCs); however, to achieve this, CSCs markers are required. This study investigated the influence of micro-environmental changes on CSCs in hypoxic, serum deprived U87-MG and the corresponding control cells. Proteomic analysis produced a wide dataset, depicting the changes that occur at the proteomic level in the differentiated and undifferentiated U87-MG cell line. With the IPA analysis, HPRD and literature reviews, 11 proteins were proposed as potential differentiated biomarkers for CSCs namely Hsp90β1, vimentin, PGK1, GAPDH, EIF4e, TPI1, HspA8, HNRNPK, NAMPT, CCSNK2A1, and ANXA2.