Amino acids are essential for life, and have many functions in metabolism. One particularly important function is to serve as the building blocks of peptides and proteins, giving rise complex three dimensional structures through disulfide bonds or crosslinked amino acids. Peptides are frequently cyclic and contain proteinogenic as well as nonproteinogenic amino acids in many instances. Since most of the amino acids contain a chiral carbon atom, the stereoisomers of all these amino acids and the peptides in which they are to be found may possess differences in biological activity in living systems. The development of methods for the separation of enantiomers has attracted great interest, since it became evident that the potential biological or pharmacological applications are mostly restricted to one of the enantiomers. The important analytical task of the separation of isomers is achieved mainly by chromatographic and electrophoretic methods. This special review surveys indirect and direct high-performance liquid chromatographic (HPLC) methods of biologically and pharmaceutically important enantiomers of nonproteinogenic amino acids and related compounds, with emphasis on the literature published from the beginning.