The medicinal value of cardiac glycoside inhibitors for the treatment of congestive heart failure symptoms stems from their ability to specifically inhibit the ion transport activity of the transmembrane enzyme sodium/potassium-ATPase (Na/K-ATPase) in myocardial cells. In this study, we used the inhibitory potencies of 39 cardiac glycoside analogues for the development of a quantitative structure-activity relationship (QSAR) model for Na/K-ATPase inhibition. In conjunction with a substructure and similarity search, the QSAR model was used to select ten potential inhibitors from a commercial compound database. The inhibitory potencies of these compounds were measured and four were found to be more active than the commonly used inhibitor ouabain. The results of the bioassays were incorporated into a second QSAR model, whose physical interpretation suggested that the nature of substituents in positions 10, 12, and 17 at the cyclopentanoperhydrophenanthrene core of the inhibitors was critical for enzyme inhibition. All descriptors of the QSAR models were conformation-independent, making the search protocol a suitable tool for the rapid virtual screening of large compound databases for novel inhibitors.