Cathepsin B, a cysteine protease, is an important target in fighting cancer. This enzyme has been implicated in enhancing tumor invasiveness and metastasis, therefore inhibitors for cathepsin B are highly sought as potential anticancer and antimetastatic agents. A structure-based design effort was pursued in arriving at a template for inhibition of cathepsin B. Focused compound libraries were synthesized based on this template, which were screened for cathepsin B inhibitory properties. Compound 2, 1-(2(R)-[1(S)-acetoxy-2-[2(S)-(2,4-difluoro-benzoylamino)-3-phenyl-propionylaminooxy]-2-oxo-ethyl]-pentanoyl)-pyrrolidine-2(S)-carboxylic acid benzyl ester, is the prototype of this novel class of cysteine protease inhibitor that emerged from the search. The molecule modifies the active site of cathepsin B covalently, irreversibly, and efficiently, a process for which the kinetic parameters were evaluated. A set of three judiciously altered variants of compound 2 was also synthesized to explore the details of the proposed mechanism of action by this inhibitor. Compound 2 and its analogues may prove useful tools in reversing the deleterious effect of cathepsin B in fighting cancer.