Cathepsin B has been found being responsible for many human diseases. Inhibitors of cathepsin B, a ubiquitous lysosomal cysteine protease, have been developed as a promising treatment for human diseases resulting from malfunction and over-expression of this enzyme. Through a high throughput screening assay, a set of compounds were found able to inhibit the enzymatic activity of cathepsin B. The binding structures of these active compounds were modeled through docking simulation. Three-dimensional (3D) quantitative structure-activity relationship (QSAR) models were constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) based on the docked structures of the compounds. Strong correlations were obtained for both CoMFA and CoMSIA models with cross-validated correlation coefficients (q²) of 0.605 and 0.605 and the regression correlation coefficients (r²) of 0.999 and 0.997, respectively. The robustness of these models was further validated using leave-one-out (LOO) method and training-test set method. The activities of eight (8) randomly selected compounds were predicted using models built from training set of compounds with prediction errors of less than 1 unit for most compounds in CoMFA and CoMSIA models. Structural features for compounds with improved activity are suggested based on the analysis of the CoMFA and CoMSIA contour maps and the property map of the protein ligand binding site. These results may help to provide better understanding of the structure-activity relationship of cathepsin B inhibitors and to facilitate lead optimization and novel inhibitor design. The multi-conformation method to build 3D QSAR is very effective approach to obtain satisfactory models with high correlation with experimental results and high prediction power for unknown compounds.