A simple and sensitive liquid chromatographic method has been developed for the determination of digoxin and digitoxin by derivatization. The method is based on the derivatization of digoxin and/or digitoxin with derivatizing agent, 1-naphthoyl chloride, in pyridine at 50?C. Under the mild condition, the digoxin and digitoxin were derivatized into their highly sensitive derivatives. After the derivatization reaction, a dimethylamine- acetonitrile solution was added to the reaction mixture to eliminate the interference from the excess derivatizing agent. The derivatives obtained were performed on a reversed-phase C8column with 88% acetonitrile as the mobile phase. The parameters affecting the derivatization of digoxin and digitoxin, including reaction temperature, reaction time, and the amount of derivatizing agent, were investigated. The linear ranges of the method for the determination of digoxin and digitoxin were over 1–50 nmol/mL and 5–50 nmol/mL; the detection limit (signal to noise ratio = 5; injection volume, 20 μL) were 0.25 nmol/mL and 0.30 nmol/mL, respectively. Partial application of the method to the analysis of digoxin in commercial tablets has proven to be satisfactory.