Digitoxin, a naturally occurring cardiac glycoside, is traditionally used for the treatment of congestive heart failure by enhancing the cardiac contraction. The mechanism of the positive cardiotonic activity was due to the increased cytosolic Ca2+ concentration from the inhibition of Na+/K+-ATPase. More recently, digitoxin is recognized for its profound anticancer activity from both epidemiological and in vitro research studies. Concern remains, however, due to the low threshold for cardiotoxicity and its narrow therapeutic window. Although there are several SAR studies conducted in the past to investigate the inhibitory interaction of cardiac glycosides against Na+/K+-ATPase in myocardial cells, but the detailed explanation of mechanism for anticancer effect is still lacking. Our main objectives are to identify novel digitoxin analogues that could selectively target the abnormality in cancer cells with stronger potency, which in turn will make drug become more effective in the presence of normal cells. We have successfully employed our Palladium-catalyzed glycosylation and post-glycosylation transformations to perform series of systematic structural-activity relationship studies on the carbohydrate portion of digitoxin against several cancers. From these studies we concluded that O-glycosides are better than N-glycosides, and that sugars with ?-D-digitoxo-, ?-L-amiceto-, and ?-L-rhamno-stereochemistry were the most active. Regardless of the glycosidic linkage (O-/N- and/or ?-/?-), the sugar stereochemistry of the monosaccharides is more effective than di- and trisaccharides. The effect of C5′-alkyl substitution on ?-L-rhamno and amiceto-sugars are found to be deleterious to cancer cell cytotoxicity. Together, these results provide significant insights toward future development of digitoxin derivative as potential anticancer agent.