Mitochondria are the leading producers of free radicals, ion homeostasis or redox state regulators, and ATP generators. They are the most critical cellular component for the endurance of aerobic cells. Numerous scientific studies have shown how vital mitochondria are to synthesizing primordial ATP, which is required for eukaryotic cells to survive and proliferate. Thus, these cell organelles are called the “powerhouse” of cell. They possess double membrane and a tiny genome. A system of bioenergetics connections that regulates a large portion of cellular activity revolves around the mitochondrion. In this regard, it is essential to comprehend the intricacy of the mitochondrial compartment and its operation and to create experimental approaches that evaluate its nature, function, and metabolism. The function, structural organization, mitochondrial respiratory chain, transmembrane potential preservation, and production of reactive oxygen species of the mitochondria are all covered in this chapter. Flow cytometry-based probes are highlighted as the primary probes to monitor mitochondrial health. A collection of methodologies for studying mitochondria is included in this chapter to facilitate the researchers in experiment designing, methodology opting tactics, analysis, and result interpretation.