The study of the mitochondrial membrane potential (ΔΨ) is essential for an integrated appraisal of mitochondrial function, since it reflects differences in electrical potential and represents the main component of the proton electrochemical gradient accounting for more than 90% of the total available respiratory energy. Numerous methods have been used to estimate mitochondrial membrane potential (ΔΨ), including fluorescent methods and electrochemical probes. In this chapter, we describe several practical approaches that allow mitochondrial membrane potential (ΔΨ) evaluation, by using a tetraphenylphosphonium (TPP⁺ )-selective electrode. The main focus is given to the evaluation of ΔΨ in isolated mitochondria.