Mitochondria are the major energy providers in most mammalian cells. Mitochondrial dysfunction has been linked to multiple diseases and pathophysiologies, including a growing number of neurodegenerative disorders. Moreover, as key regulators of cell death, mitochondria are also prime targets for cancer research and at the center of drug development. The mitochondrial membrane potential is essential for ATP production and an important parameter to assess the functional state of these organelles. As MitoTracker® dyes concentrate in active mitochondria, they are ideal tools to evaluate mitochondrial function. We have developed a simple, fast and reliable protocol that measures MitoTracker® signals associated with mitochondria. Our protocol was successfully applied to assess changes in the mitochondrial membrane potential in diverse cell types from different organisms. The method developed by us provides a powerful tool for mitochondrial research and can be easily adapted for drug screening.