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In assisted reproduction, ejaculated semen is either washed immediately for in vitro fertilization or diluted and processed further for optimal low temperature liquid- or cryo-preservation. The extended handling of semen makes sperm vulnerable to organelle and/or membrane damages. In addition to several important functions, mitochondrion is chiefly responsible for production of detrimental free radicals in spermatozoa. Therefore, direct evaluation of mitochondrial activitymightbe useful in semen evaluation protocol as an altate, more objective and desirable parameter of spermatozoa quality. This is further aided by availability of wide spectral range of fluorescent probes that has advantage of simultaneous multi-parametric assay. Even with availability of several mitochondrial-specific probes, it is sometimes difficult for an investigator to select a most suited dye for experimentation. Therefore, in this review the literature pertaining to role of the mitochondria in the sperm cell, focusing on morphology, maintenance of trans-membrane potential, mechanism involved in generation of reactive species, and comparative analysis of a number of probes used for evaluating mitochondrial function in sperm cells are discussed. Our intention is to present concise information on the technical aspects of various probing methods, and this might be useful for investigators to design experimental approach by proper selection of the dye and for accurate interpretation of the results.