This review discusses the synthesis, mechanisms of action, and therapeutic and diagnostic applications of mitochondria-targeted triphenylphosphonium (TPP*) compounds. Mitochondria are crucial targets for drug development in cancer, cardiovascular, and neurological diseases. The most effective method to deliver drugs specifically to mitochondria is by covalently linking a lipophilic cation, such as an alkyltriphenylphosphonium moiety, to the pharmacophore of interest. Other lipophilic cations, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles have also been used for mitochondrial delivery. The accumulation of lipophilic cations in mitochondria is driven by the mitochondrial membrane potential, with over 1000-fold higher mitochondrial concentration achievable. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and to treat various diseases. This review covers the physicochemical basis for mitochondrial accumulation, synthetic strategies, mitochondrial probes, and sensors, as well as examples of bioactive compounds. It also reviews the application of mitochondria-targeted probes for in vivo imaging of myocardial function and tumors. The advantages of TPP*-based mitochondrial targeting include stability, lipophilic and hydrophilic properties, simple synthesis, and low reactivity. The review highlights the potential of mitochondria-targeted compounds in treating various diseases and their clinical significance.This review discusses the synthesis, mechanisms of action, and therapeutic and diagnostic applications of mitochondria-targeted triphenylphosphonium (TPP*) compounds. Mitochondria are crucial targets for drug development in cancer, cardiovascular, and neurological diseases. The most effective method to deliver drugs specifically to mitochondria is by covalently linking a lipophilic cation, such as an alkyltriphenylphosphonium moiety, to the pharmacophore of interest. Other lipophilic cations, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles have also been used for mitochondrial delivery. The accumulation of lipophilic cations in mitochondria is driven by the mitochondrial membrane potential, with over 1000-fold higher mitochondrial concentration achievable. Mitochondrial targeting has been developed to study mitochondrial physiology and dysfunction and to treat various diseases. This review covers the physicochemical basis for mitochondrial accumulation, synthetic strategies, mitochondrial probes, and sensors, as well as examples of bioactive compounds. It also reviews the application of mitochondria-targeted probes for in vivo imaging of myocardial function and tumors. The advantages of TPP*-based mitochondrial targeting include stability, lipophilic and hydrophilic properties, simple synthesis, and low reactivity. The review highlights the potential of mitochondria-targeted compounds in treating various diseases and their clinical significance.