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Mitochondria are organelles with significant cellular functions, especially cellular bioenergetics and apoptosis. They are structural and functional elements of cell respiration with the electron transport system (ETS), whose role is to provide adenosine triphosphate (ATP), used as a source of chemical energy. The Krebs cycle and fatty acid oxidation take place within mitochondria. Other metabolic pathways and cycles include some steps inside and outside the mitochondria (e.g., the urea cycle, steroid biosynthesis, heme biosynthesis, and cardiolipin synthesis). Dysfunction of mitochondria plays a critical role in the pathophysiology of a variety of diseases including degenerative diseases, aging, and cancer, etc. Nowadays the interest of the mitochondrial respiratory function is still increasing due to their importance in the physiology and pathophysiology of an organism. Neuroblastoma cell line SH-SY5Y is widely used as an in vitro model in neurodegenerative diseases, where mitochondrial dysfunction is considered as a key mechanism in pathophysiology of neurodegenerative disorders. This paper gives first insight into the mitochondrial respiration and characterization of SH-SY5Y cells, with basic information of respiration in different coupling control states including ROUTINE, LEAK, and maximal electron transport (ET) capacity.