The mitochondrial functions perform the energy required to support cell activities. The mitochondrial oxidative phosphorylation system attains the function of a powerhouse by energy production in the form of ATP. However, calcium and iron homeostasis, intermediate metabolism, biosynthesis of steroids, and heme are processes exploited in the mitochondria coupled to uptake and efflux of substrates through mitochondrial membranes. Moreover, in turn, oxidative cellular energy metabolism is influenced by mitochondrial dynamics and morphology. The physiological mitochondrial cycle to maintain the homeostasis of biogenesis/mitophagy or fusion/fission dynamics transformations of mitochondria prevents impaired cell bioenergetics. However, mitochondrial dysfunction and the phenomenon of mitochondrial permeability transition pore trigger the biological phenomena responsible for different forms of regulated cell death. This special issue aims to describe the most relevant laboratory techniques used to promote scientific research and teaching on mitochondrial studies.