The mitochondrion performs critical roles in eukaryotic cells including ATP production, cell growth, survival, apoptosis, and differentiation. Many human diseases can be traced to dysfunction within the mitochondria, but selective delivery of therapeutics into the mitochondria has been challenging. This chapter describes the detailed protocols for the synthesis of a new family of mitochondrion-targeting, cell-penetrating molecules (CPMs) and their application for the delivery of small-molecule and peptidyl cargos into the mitochondrial matrix. Live-cell confocal microscopic imaging of HeLa cells treated with a variety of CPM-cargo conjugates revealed that the CPMs efficiently and specifically deliver membrane-impermeable linear and cyclic peptidyl cargos into the mitochondrial matrix, as long as the cargo carries no more than two negative charges.