Myocardial infarction (MI), cardiac ischemia and consequent reperfusion injury (I/R), are among the most significant triggers of pathophysiological oxidative stress of the myocardium and correlated with unfavorable metabolic perturbations; yet our knowledge regarding cross-talk between redox stress and cardiac metabolism remains limited. Here we aimed to unravel key oxidative and metabolic events during the early onset of oxidative stress induced cardiac damage. To that end, we simulated I/R in stem-cell derived, metabolically matured cardioids as well as carefully collected cardiac tissues of mice subjected to MI. The samples were processed with our established one pot, two-step alkylation redox approach, covering protein oxidation, protein abundance, as well as redox state of small thiols. Of note, the data from both I/R cardioids and MI tissues were compared to their corresponding non-stressed controls. As a result, we demonstrate that both in vitro and in vivo I/R affected a number of metabolic enzymes, especially those involved in (mitochondrial) sugar and lipid catabolism and lipid and amino acid biosynthesis/uptake. In addition, we report significant perturbations of cellular thiolome across the two model systems, manifested by prominent protein oxidation (especially of mitochondrial proteins) and increase in cysteine levels and glutathione synthesis. Tightly related to glutathione, in the core of the overlap between proteome signatures of in vitro and in vivo I/R we identified the upregulation of serine uptake and its de novo synthesis. Correspondingly, tissue serine levels were increased in MI-affected hearts compared to sham control, both in areas directly affected by MI as well as MI distal zones of the same hearts. We explored this demand for serine and can report that serine availability influences cellular apoptosis during and post I/R as well as glutathione production, suggesting vital role of serine in I/R injury. Collectively, our data emphasizes oxidation vulnerability of mitochondria during cardiac I/R and highlights serine as one of the key players implicated in oxidative stress amelioration during and upon cardiac oxidative injury.