Introduction: Metabolic remodelling and oxidative stress are central contributors to heart disease progression, yet disentangling causative mechanisms from downstream effects remains challenging. Prior analyses on failing human hearts and murine tissue have implicated redox homeostasis as a key determinant of cardiac pathology, including the regulation of metabolic flux through enzymes such as pyruvate kinase M (PKM). Traditional models often lack physiological fidelity, limiting translational insights. Advanced cardiac organoids (cardioids) provide an in vitro platform that retains critical in vivo-like characteristics, enabling a more precise examination of redox-metabolic interactions in clinically relevant settings. Here, we present a metabolically matured cardioid system optimised to dissect ischemia-reperfusion injury (RI)-induced perturbations of the cellular metabolic and redox landscape. Methods: To comprehensively investigate the interplay between aberrant metabolism and disturbed redox environments, a one-pot multiomics workflow integrating global proteomics, redox proteomics, and targeted redox metabolomics was employed. State-of-the-art mass spectrometry, combined with preservative two-step cysteine alkylation, enabled the precise assessment of glutathione redox status and its derivatives as proxies for cellular oxidative stress and antioxidant capacity. Additionally, capturing site-specific information of individual redox-sensitive (cysteine-containing) peptides provided high-resolution profiling of oxidative modifications, complementing proteomic analysis and offering more profound insights into redox-dependent signalling. A novel human induced pluripotent stem cell (hiPSC)-derived cardiac organoid model was refined through a metabolic maturation protocol and subsequently characterised. The matured model was then subjected to normoxia, hypoxia, and ischemia-reperfusion conditions. To further elucidate the putative role of PKM, pharmacological activation via TEPP-46, a selective allosteric activator of PKM2, was concurrently incorporated into the experimental setup. Results: The matured cardioid model exhibited enhanced (oxidative) metabolism, mitochondrial organisation and function, and structural fidelity, as indicated by changes in the cellular proteome and redox status, making it particularly suited for studying the intersection of metabolic dysfunction and redox imbalance in cardiac disease states. Building on this, we offer a comprehensive multiomics-based depiction of the model’s response in pathophysiologically relevant conditions, revealing distinct molecular signatures that differentiate ischemia-reperfusion from hypoxia-only conditions and highlighting unique vulnerabilities in RI. Additionally, TEPP-46-mediated PKM2 activation under corresponding conditions provided further insights into its role as a key regulator of glycolytic flux and redox homeostasis, reinforcing previous findings in failing heart tissue. Conclusions: This study establishes an optimised cardioid platform and mass spectrometry-based multiomics as a robust approach for investigating redox-regulated metabolic dysfunction in ischemia-reperfusion injury. Our findings support previously reported alterations in myocardial redox homeostasis in heart failure and identify redox-sensitive enzymes such as PKM as critical metabolic control nodes and potential therapeutic targets in cardiac stress responses. Furthermore, the ability to delineate RI-specific effects from hypoxia-driven changes further underscores the translational relevance of a systematic approach in modelling cardiac pathology and exploring targeted cardioprotective strategies.