Proper regulation of neuronal gene expression is crucial for the development and differentiation of the central nervous system. The transcriptional repressor REST (repressor element-1 silencing transcription factor) is a key regulator in differentiation of pluripotent stem cells to neuronal progenitors and mature neurons. Dysregulated REST activity has been implicated in various diseases, among which the most deadly is glioblastoma multiforme (GBM). Here we have developed an expression-based REST signature (EXPREST), a device providing quantitative measurements of REST activity for GBM tumors. EXPREST robustly quantifies REST activity (REST score) using gene expression profiles in absence of clinic-pathologic assessments of REST. Molecular characterization of REST activity identified global alterations at the DNA, RNA, protein and microRNA levels, suggesting a widespread role of REST in GBM tumorigenesis. Although originally aimed to capture REST activity, REST score was found to be a prognostic factor for overall survival. Further, cell lines with enhanced REST activity was found to be more sensitive to IGF1R, VEGFR and ABL inhibitors. In contrast, cell lines with low REST score were more sensitive to cytotoxic drugs including Mitomycin, Camptothecin and Cisplatin. Together, our work suggests that therapeutic targeting of REST provides a promising opportunity for GBM treatment.