Mitochondria are essential organelles in cells, where they generate ATP, synthesize metabolites, and buffer calcium. These organelles have dynamic properties that respond to metabolic demands within cellular subcompartments or external stimuli to fulfill critical functions. Molecular components regulate their morphology, clearance, contact with organelles, and transport. Neurons, which have long axons that are sensitive to mitochondrial dysfunction, regard transport as critically important because it is often associated with the pathogenesis of neurodegenerative disorders. The polarized shape of neurons has been utilized to study the dynamics of mitochondria across species and at different stages of development. The mechanisms that occur in neurons have also been observed in other cellular types and intracellular transport. This Methods Collection aims to provide crucial tools for studying mitochondrial transport in vivo and in vitro.