This review discusses the synthesis, mechanisms of action, and therapeutic and diagnostic applications of mitochondria-targeted triphenylphosphonium (TPP⁺)-based compounds. Mitochondria are important targets for drug design in cancer, cardiovascular, and neurological diseases. The most effective way to deliver drugs to mitochondria is by covalently linking a lipophilic cation, such as an alkyltriphenylphosphonium moiety, to a pharmacophore. Other lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach and membrane potentials, mitochondrial concentrations can be up to 1000-fold higher. Mitochondrial targeting has been used to study mitochondrial physiology and dysfunction, and for treating diseases like neurodegeneration and cancer. The review discusses efforts to target small-molecule compounds to mitochondria for probing mitochondrial function, as diagnostic tools and potential therapeutics. It describes the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic strategies for targeting compounds to mitochondria, mitochondrial probes and sensors, and examples of mitochondrial targeting of bioactive compounds. It also reviews published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases. The review highlights the advantages of TPP⁺-based mitochondrial targeting, including stability in biological systems, lipophilic and hydrophilic properties, simple synthesis and purification, low chemical reactivity, and lack of light absorption or fluorescence. The review also discusses other approaches to target compounds to mitochondria, including linking to heterocyclic aromatic cations, mitochondria-targeted peptides, and mitochondria-targeted vesicles. The review also discusses the transport of small cationic compounds and biomolecules to mitochondria, the structure of the mitochondrial membrane, the effect of charge and hydrophobicity of the compound, and the effect of protonation equilibria of weak acids and bases on their mitochondrial accumulation. The review also discusses mitochondrial labeling compounds and membrane potential indicators, the effect of mitochondria-targeted compounds on mitochondrial respiration, and synthetic approaches to mitochondria-targeted compounds. The review concludes that mitochondria-targeted compounds have significant potential for therapeutic and diagnostic applications in various diseases.This review discusses the synthesis, mechanisms of action, and therapeutic and diagnostic applications of mitochondria-targeted triphenylphosphonium (TPP⁺)-based compounds. Mitochondria are important targets for drug design in cancer, cardiovascular, and neurological diseases. The most effective way to deliver drugs to mitochondria is by covalently linking a lipophilic cation, such as an alkyltriphenylphosphonium moiety, to a pharmacophore. Other lipophilic cations, such as rhodamine, natural and synthetic mitochondria-targeting peptides, and nanoparticle vehicles, have also been used for mitochondrial delivery of small molecules. Depending on the approach and membrane potentials, mitochondrial concentrations can be up to 1000-fold higher. Mitochondrial targeting has been used to study mitochondrial physiology and dysfunction, and for treating diseases like neurodegeneration and cancer. The review discusses efforts to target small-molecule compounds to mitochondria for probing mitochondrial function, as diagnostic tools and potential therapeutics. It describes the physicochemical basis for mitochondrial accumulation of lipophilic cations, synthetic strategies for targeting compounds to mitochondria, mitochondrial probes and sensors, and examples of mitochondrial targeting of bioactive compounds. It also reviews published attempts to apply mitochondria-targeted agents for the treatment of cancer and neurodegenerative diseases. The review highlights the advantages of TPP⁺-based mitochondrial targeting, including stability in biological systems, lipophilic and hydrophilic properties, simple synthesis and purification, low chemical reactivity, and lack of light absorption or fluorescence. The review also discusses other approaches to target compounds to mitochondria, including linking to heterocyclic aromatic cations, mitochondria-targeted peptides, and mitochondria-targeted vesicles. The review also discusses the transport of small cationic compounds and biomolecules to mitochondria, the structure of the mitochondrial membrane, the effect of charge and hydrophobicity of the compound, and the effect of protonation equilibria of weak acids and bases on their mitochondrial accumulation. The review also discusses mitochondrial labeling compounds and membrane potential indicators, the effect of mitochondria-targeted compounds on mitochondrial respiration, and synthetic approaches to mitochondria-targeted compounds. The review concludes that mitochondria-targeted compounds have significant potential for therapeutic and diagnostic applications in various diseases.