Cell-penetrating peptides (CPPs) have found numerous applications in biology and medicine since the first synthetic cell-permeable sequence was identified two decades ago. Numerous types of drugs have been transported into cells using CPPs, including small-molecule pharmaceuticals, therapeutic proteins, and antisense oligonucleotides. Improved agents for medical imaging have been generated by conjugation with CPPs, with the appended peptides promoting cellular uptake and in some cases, cell-type specificity. Organelle-specific CPPs have also been generated, providing a means to target specific subcellular sites. This review highlights achievements in this area and illustrates the numerous examples where peptide chemistry was exploited as a means to provide new tools for biology and medicine.