Background: NGS is becoming increasingly available but its relevance in clinical practice has been questioned. In this prospective, IRB-approved study (NCT01775072), we utilized the MSK-IMPACT assay to analyze gliomas in a routine practice setting. The MSK-IMPACT, performed in a CLIA-compliant laboratory, is a multiplexed assay (Illumina HiSeq) providing full exon coverage of 341 cancer related genes, detecting base substitutions, small indels, copy number and select gene rearrangements. Methods: After written consent, tumor and germline DNA were analyzed. Mutations were catalogued and compared to TCGA data. Comparison with other profiling methods, i.e. IHC (IDH-1 R132H mutation), RT-PCR (EGFRvIII), Sanger sequencing (IDH-1/2 mutations) and FISH (EGFR amplification, 1p/19q co-deletion) was performed. Participation in clinical trials was recorded. Results: N = 104 pts were enrolled (48 glioblastomas, 39 grade III, 9 grade II and 3 grade I gliomas). The mutational landscape was in line with TCGA, including mutations in TERT (59%), TP53 (55%), IDH-1 (35%), ATRX (27%), PTEN (22%) EGFR (17%), PIK3CA (12%) and BRAF (2%); four pts displayed a hypermutator genotype. MSK-IMPACT identified all IDH1/2 mutations, including one IDH2 R172K in an IDH-1 R132H IHC-negative tumor. All pure oligodendrogliomas (N = 13) displayed 1p/19q codeletion on MSK-IMPACT. Three pts had 1p/19q codeletion on FISH but not on MSK-IMPACT, but mutational profile/ histology favored non-codeleted glioma, suggesting false-positive FISH results. All FISH EGFR amplifications were detected by MSK-IMPACT. Among EGFR amplified tumors, MSK-IMPACT disclosed EGFRvIII in 55%. To date, 33 pts have been enrolled in trials, including 4 extreme responders in whom results are guiding further drug development. Conclusions: NGS with MSK-IMPACT is a highly useful profiling tool, providing prognostic and therapeutically relevant information and guiding patient selection/ interpretation of clinical trials. Given lower cost, higher accuracy and wider range of information provided, this assay replaces with advantages other profiling tools, and is ready for incorporation into routine clinical practice. Clinical trial information: NCT01775072