Background Cancer cells use multiple mechanisms to counteract oxidative stress. Reactive oxygen species can oxidatively modify enzymes to reroute metabolic pathways according to tumor needs, e.g., for antioxidant production/recycling. Therefore, identification of oxidized proteins is an important step for understanding mechanisms that drive cancer cell survival. Thiol groups are most susceptible to oxidative modifications but rarely analyzed in clinical settings due to their reactivity and associated post-sampling artifacts. Methods To accurately address the cross-talk between redox signaling and metabolism we collected tumor and healthy tissue from 70 individuals with non-small cell lung cancer right after surgery into a thiol-quenching solution, then carried out redox-proteomics and small molecular thiol analysis. Results and conclusions As a result of such an unbiased approach, we for the first-time show evidence of higher oxidation of a number of proteins linked to pro-oncogenic pathways including key metabolic enzymes in human tumors (especially glucose-related). We demonstrate that cancer adapts metabolism to increase glutathione synthesis to fight the rise of intracellular oxidative stress. Glyoxalases, the key enzymes in the detoxification of methylglyoxal, a byproduct of glycolysis and precursor of advanced glycation end products, were compromised by oxidative modifications and downregulation on protein level. Despite decreased methylglyoxal detoxification capacity, however, cancers did not accumulate advanced glycation end products. Since downregulation or inhibition of GAPDH activity led to upregulation of glyoxalases in non-small cell lung cancer cells we propose that tumors efficiently avoid methylglyoxal formation by upregulation of GAPDH activity.