They are essential for normal cell function by providing energy through their role in oxidizing carbon substrates. Glutathione (GSH) is a major thiol-disulfide redox buffer of the cell including the mitochondrial matrix and intermembrane space. We have generated cardiomyocyte-specific Grx1-roGFP2 GSH redox potential (E GSH ) biosensor mice in the past, in which the sensor is targeted to the mitochondrial matrix. Using this mouse model a distinct E GSH of the mitochondrial matrix (−278.9 ± 0.4 mV) in isolated cardiomyocytes is observed. When analyzing the E GSH in isolated mitochondria from the transgenic hearts, however, the E GSH in the mitochondrial matrix is significantly oxidized (−247.7 ± 8.7 mV). This is prevented by adding N-Ethylmaleimide during the mitochondria isolation procedure, which precludes disulfide bond formation. A similar reducing effect is observed by isolating mitochondria in hypoxic (0.1–3% O 2 ) conditions that mimics mitochondrial pO 2 levels in cellulo. The reduced E GSH is accompanied by lower ROS production, reduced complex III activity but increased ATP levels produced at baseline and after stimulation with succinate/ADP. Altogether, we demonstrate that oxygenation is an essential factor that needs to be considered when analyzing mitochondrial function ex vivo.