Mitochondria, the complex powerhouses of eukaryotic cells, lie at the core of energy production, metabolism, and signaling. Mitochondrial dysfunctions underlie a wide range of human diseases, and there is a need for simple and effective tools to target and study these organelles. This review focuses on the applications of mitochondria-targeted cationic probes. It provides an up-to-date review of recent publications investigating the effects of these cationic probes, which are designed to manipulate mitochondrial function and detect dysfunction in different cell lines. In addition, it analyzes the effects of mitochondria-targeted fluorescence cationic probes in vivo and in vitro studies, and their effects in probe studies.