In this article, we describe the development and validation of a custom targeted NGS approach for the analysis of 13 glioma-related genes (ACVR1, ATRX, BRAF, CDKN2A, EGFR, H3F3A, HIST1H3B, HIST1H3C, IDH1, IDH2, P53, PDGFRA, PTEN), a 125 bp region of the TERT promoter (TERTp), and 54 single nucleotide polymorphisms (SNPs) located along chromosomes 1 and 19 for the reliable assessment of their copy number alterations (CNAs). This evidence concerns the gene H3-3A and glioma.