The field of mitochondrial biochemistry has developed rapidly in recent years, especially in the areas of electron transport chain, mitochondrial oxidative phosphorylation and metabolomics related studies, and many milestones have been achieved. Mitochondria are the centre of cellular energy metabolism, participating in fatty acid oxidation, tricarboxylic acid cycle, electron transport chain, oxidative phosphorylation, and converting chemical energy from ingested food nutrients into the cellular universal energy carrier ATP through oxidation-reduction reactions, providing the main source of energy for the cell. However, the increased production of abnormal ROS in mitochondria and the accumulation of oxidative stress will lead to a decrease in mitochondrial quality, bioenergetic dysfunction and metabolic stress, which in turn induce many human diseases. We will create a special issue to assess mitochondrial structure, function, mtDNA, metabolism and bioinformatics methodologies to provide a framework for the practical assessment of mitochondrial quality. This collection aims to provide protocols and guidelines for the development and application of different biopsy methods. It will be of interest to researchers studying various physiological and pathological phenomena in which decreased mitochondrial quality is a contributing factor.