© Springer Science+Business Media New York 2015. Mitochondria play key roles in physiology and in disease. Understanding the mechanisms that defi ne mitochondrial function, the regulation of energy balance in the cell, and the ways in which mitochondrial dysfunction impacts on cell physiology represents a key challenge in modern cellular pathophysiology. This requires a study of mitochondrial function in relation to the physiology of the cell, and so has driven approaches to measure and characterize key aspects of mitochondrial function within living cells. Central to this advance has been the use of fluorescent indicators that report mitochondrial membrane potential, mitochondrial redox state, rates of free radical generation, ATP, and so on. In this chapter we provide a critical appraisal and guide to the measurement of mitochondrial bioenergetics within living cells and discuss both the strengths and potential pitfalls of the most useful probes for mitochondrial membrane potential using confocal microscopy and flow cytometry.