The mitochondrial membrane potential (ΔΨm) is a critical component in energy storage during oxidative phosphorylation, formed by proton pumps (Complexes I, III, and IV). ΔΨm, along with the proton gradient (ΔpH), creates a transmembrane potential of hydrogen ions that drives ATP synthesis. The levels of ΔΨm and ATP are maintained within a stable range, but sustained changes can be detrimental. ΔΨm plays a key role in mitochondrial homeostasis, including the selective elimination of dysfunctional mitochondria through mitophagy. It also drives the transport of ions and proteins necessary for mitochondrial function. The paper discusses the significance of ΔΨm in cellular health and viability, and provides recommendations for accurate measurement methods, highlighting potential artifacts. The authors explore the mechanisms by which ΔΨm is essential for various cellular processes, such as protein transport, retrograde signaling, and the regulation of mitochondrial ROS production. They also discuss the heterogeneity of ΔΨm and its implications for pathologies, emphasizing the importance of mitochondrial quality control. Finally, they review the challenges and limitations of measuring ΔΨm using fluorescent probes, suggesting TMRM as a reasonable compromise under controlled conditions.The mitochondrial membrane potential (ΔΨm) is a critical component in energy storage during oxidative phosphorylation, formed by proton pumps (Complexes I, III, and IV). ΔΨm, along with the proton gradient (ΔpH), creates a transmembrane potential of hydrogen ions that drives ATP synthesis. The levels of ΔΨm and ATP are maintained within a stable range, but sustained changes can be detrimental. ΔΨm plays a key role in mitochondrial homeostasis, including the selective elimination of dysfunctional mitochondria through mitophagy. It also drives the transport of ions and proteins necessary for mitochondrial function. The paper discusses the significance of ΔΨm in cellular health and viability, and provides recommendations for accurate measurement methods, highlighting potential artifacts. The authors explore the mechanisms by which ΔΨm is essential for various cellular processes, such as protein transport, retrograde signaling, and the regulation of mitochondrial ROS production. They also discuss the heterogeneity of ΔΨm and its implications for pathologies, emphasizing the importance of mitochondrial quality control. Finally, they review the challenges and limitations of measuring ΔΨm using fluorescent probes, suggesting TMRM as a reasonable compromise under controlled conditions.