The major component of mitochondrial electrochemical potential gradient of protons is the mitochondrial membrane potential (DeltaPsi), and hence it is a suitable parameter for assessment of mitochondrial function. Dissipation of the mitochondrial membrane potential causes uncoupling of the electron transport through the respiratory chain and the phosphorylation reaction for ATP synthesis (proton leak). Proton leak functions as a regulator of mitochondrial reactive oxygen species (ROS) production and its modulation by uncoupling proteins, which may be involved in pathophysiology. In this report, we describe the assays for mitochondrial membrane potential and proton leak, which require a TPP(+) electrode and a Clark electrode. The determination of mitochondrial peroxide production with homovanillic acid is also included.