Abstract: Digitalis glycosides of cardenolide group are important constituents with closely similar structures isolated from Digitalis plants of Sreophulariaceae for the treatment of heart diseases. A reversed-phase HPLC has been developed to separate 13 digitalis glycosides successfully by using methanol as a modifier to acetonitrile-water in concave gradient elution from (A) CH₃CN-H₂O-MeOH (30:70:10) to (B) CH₃CN-H₂O-MeOH (52.5: 47.5: 12). The effects of methanol on the separation of the digitalis glycosides and the characteristics between retention time and structures are also hereby discussed.