Mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian cells. The generation of ROS underlies many physiological and pathophysiological processes that occur within cellular systems. Superoxide ([image omitted] ) is the proximal ROS generated during electron 'leakage' from the mitochondrial electron transport chain (mETC) and is known to be released at mitochondrial complex I and complex III. Monitoring mitochondrial [image omitted] production directly and in real-time offers the potential to improve understanding of the complex mechanisms involved during mitochondrial [image omitted] generation. This study reports the novel application of a cytochrome c functionalized amperometric sensor for monitoring [image omitted] generation in isolated mitochondrial fractions. The non-invasive sensor system described allowed a comparison of [image omitted] production following specific inhibition of complex I and complex III of the mETC to be made directly and in real-time.