Gliomas are the most common primary brain tumors of adults, with a yearly incidence of 25000 cases in Unites States. The most common form of glioma is the glioblastoma multiforme (GBM) grade IV, an aggressive and malignant tumor. Glioblastomas can either develop in a rapid de novo manner (primary) or by secondary progression from less malignant glioma (astrocytoma grade II or III). The aim of the present work is the development of 2D gel electrophoresis for protein separation and mass spectrometry analysis from very limited amount of complex protein mixture. It should pave the way to analyze proteins extracted from brain tumors which have limited amount of proteins. To develop a proteomic approach, we used a cell line T98G derived from GBM. Proteins were extracted from cells in electrophoresis buffer (7.7M urea, 2.2M thiourea, 4.4M CHAPS and protease inhibitor cocktail). Extract of proteins contained 100 (A), 200 (B) and 400 ug (C) were submitted to 2D gel using IPG pH 3-10NL (7cm) for isoelectric focusing and 12% SDS-PAGE mini-gel as second dimension. Gels were stained with silver stain (mass spectrometry compatible) and colloidal coomassie blue (CCB), and no significant difference were noted in spot detection. 2D gels were scanned and analyzed for reproducibility, spot detection and spot intensity correlation using the ImageMaster 2D Platinum v.5.0 software. 2D spots were hydrolyzed by trypsin and peptides were analyzed by MALDI-TOF-MS after desalting in POROS R2. Sixty percent of spots were identified based on peptide monoisotopic masses searched against NCBI for protein identification using MS-FIT algorithm from gels B and C. The averaged number of spots detected in gel A was 237, gel B 298 and gel C 353 spots with 91.36% of reproducibility of spot location between these three gels. Analysis of staining variation of the same spot in three gels were based on intensity of CCB stain, and it gave the linear correlation of 0.97± 0.04 (n=15) in good agreement with the amount of proteins applied to gels. The MS spectra quality was evaluated by signal to noise ratio which was 2.8 for gel A (100ug), 4.5 for gel B (200ug) and 35.9 for gel C (400ug). These results taken together indicated that the minimum amount of 200ug of protein were able to provide unambiguously protein spot detection and also protein identification and will be used for tumors brain studies. FAPESP- CTC-CEPID.