Dendrimers have emerged as promising carriers for the delivery of a wide variety of pay-loads including therapeutic drugs, imaging agents and nucleic acid materials to biological systems. The current work aimed to develop a novel mitochondria-targeted generation 5 poly(amidoamine) (PAMAM) dendrimer (G(5)-D). To achieve this goal, a known mitochondriotropic ligand triphenylphosphonium (TPP) was conjugated with the surface of the dendrimer. A fraction of the cationic surface charge of G(5)-D was neutralized by partial acetylation of the primary amine groups. Next, the mitochondria-targeted dendrimer was synthesized via the acid-amine-coupling conjugation reaction between the acid group of (3-carboxypropyl)triphenyl-phosphonium bromide and the primary amines of the acetylated dendrimer (G(5)-D-Ac). These dendrimers were fluorescently labeled with fluorescein isothiocyanate (FITC) to quantify cell association by flow cytometry and for visualization under confocal laser scanning microscopy to assess the mitochondrial targeting in vitro. The newly developed TPP-anchored dendrimer (G(5)-D-Ac-TPP) was efficiently taken up by the cells and demonstrated good mitochondrial targeting. In vitro experiments carried out on normal mouse fibroblast cells (NIH-3T3) demonstrated low cytotoxicity of the G(5)-D-Ac-TPP. This mitochondria-targeted dendrimer-based nanocarrier could be useful for selective delivery of bio-actives to the mitochondria for imaging and treatment of diseases associated with mitochondrial dysfunction.