The production of reactive oxygen species (ROS) has been implicated for numerous pathologic alterations, including neurodegeneration and aging. They are formed to a considerable extent by mitochondria by single electron reduction of molecular oxygen by competent electron donors like flavoproteins and semiubiqunone species. In this chapter, we evaluate quantitative methods for the detection of hydrogen peroxide and superoxide production. Applying these methods we compared the ROS production of isolated mitochondria of mouse brain and skeletal muscle. We substantiated previous evidence that most mitochondrial ROS are produced at complexes I and III of the respiratory chain and that the contribution of individual complexes to ROS production is tissue dependent.