Mitochondria from affected tissues of Amyotrophic Lateral Sclerosis (ALS) patients show morphological and biochemical abnormalities. The impairment of mitochondrial functions causes oxidative damage and accumulation of ROS and represents one of the major features that lead to the selective death of motor neurons in ALS. Our aim was to assess the ability of oxidative stress found in ALS to induce post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific post-translational modifications (PTMs) of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neurons model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS. Selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1 or NSC34wt cells were found. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not fromNSC34control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons.