Glioblastoma Multiforme is a cancer type with an important mitochondrial component. Here was used mitochondrial proteome Random Sampling in 2D gels from T98G (oxidative metabolism) and U87MG (glycolytic metabolism) cell lines to obtain and analyze representative spots (regardless of their intensity, size, or difference in abundance between cell lines) by Principal Component Analysis for protein identification. Identified proteins were ordered into specific Protein-Protein Interaction networks, to each cell line, showing mitochondrial processes related to metabolic change, invasion, and metastasis; and other nonmitochondrial processes such as DNA translation, chaperone response, and autophagy in gliomas. T98G and U87MG cell lines were used as glioblastoma transition model; representative proteomic signatures, with the most important biological processes in each cell line, were defined. This pipeline analysis describes the metabolic status of each line and defines clear mitochondria performance differences for distinct glioblastoma stages, introducing a new useful strategy for the understanding of glioblastoma carcinogenesis formation.Biological significanceThis study defines the mitochondria as an organelle that follows and senses the carcinogenesis process by an original proteomic approach, a random sampling in 2DE gels to obtain a representative spots sample and analyzing their relative abundance by Principal Components Analysis; to faithfully describe glioblastoma cells biology.