An analytical method for the determination of cardiac glycosides in Digitalis lanata leaves by reversed-phase thin-layer chromatography (RP-TLC) was developed. The procedure consisted of extraction of dry leaf powder with 50% methanol and clean-up by Sep-Pak cartridges prior to RP-TLC analysis. RP-TLC was performed on an octadecylsilyl bonded silica gel plate, using a developing solvent of acetonitrile-methanol-0.5 M NaCl (1:1:1) for primary glycosides and acetonitrile-methanol-0.5 M NaCl (12:7:9) for secondary glycosides. The plate was scanned with a reflectance densitometer at 225 nm. The quantitation was carried out by the internal standard method. The present method is reliable and relatively simple for the determination of cardiac glycosides in Digitalis lanata leaves.