A rapid and versatile series of high-performance liquid chromatographic systems are described for the resolution of digoxin, digitoxin and their potential metabolites or degradation products and impurities. These systems consist of isocratic, single-step gradient and linear gradient modes that provide resolution of the glycosides in 25, 17 and 14 min respectively. Digoxin, its mono- and bisdigitoxosides, digoxigenin and gitoxin, a potential impurity, may be isocratically separated in 11 min. The two semi-synthetic glycosides alpha- and beta-acetyldigoxin are resolved and separated from digoxin and its metabolites in a chromatographic time of 23 min. Digitoxin and its metabolites or degradation products may be separated in as little as 9 min using an isocratic system. The solvent systems employ varying proportions of methanol, water, isopropanol and dichloromethane and a conventional 5 micrometers bonded, octadecyl phase. Detection was accompanied using a variable wavelength detector set at 220 nm.