The prognosis of patients with glioblastoma multiforme, the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation, and chemotherapy. Genetic heterogeneity of glioblastoma warrants extensive studies to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. In the present study, we report a novel proteomic approach by using two-dimensional difference gel electrophoresis followed by spot picking and analysis of proteins/peptides by Mass spectrometry. We report at least ten different novel proteins/peptides as identified by this technique which are differentially expressed in this cancer and could be of further importance for diagnostic, therapeutic, and prognostic approaches.