The proton electrochemical gradient generated by the respiratory chain activity accounts for over 90% of the available respiratory energy and, as such, its evaluation and accurate measurement regarding total values and fluctuations are an invaluable component of the understanding of mitochondrial function. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulation and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. In this chapter, we describe how Δψ may be evaluated in isolated mitochondria and live cells using electrochemical and fluorescent methods, such as tetraphenylphosphonium (TPP+) and tetramethylrhodamine methyl ester (TMRM), respectively. These methods are dependent on the accumulation of cationic probes within mitochondria, which are assessed by using a TPP+-selective electrode or instruments that measure fluorescence (microplate reader and flow cytometer).