AbstractChagas disease, caused by the protozoan Trypanosoma cruzi, is one of the most serious amongst the so‐called neglected diseases in Latin America, specially in Brazil. So far there has been no effective treatment for the chronic phase of this disease. Cruzain is a major cysteine protease of T. cruzi and it is recognized as a valid target for Chagas disease chemotherapy. The mechanism of cruzain action is associated with the nucleophilic attack of an activated sulfur atom towards electrophilic groups. In this report, features of a putative pharmacophore model of the enzyme, developed as a virtual screening tool for the selection of potential cruzain inhibitors, are described. The final proposed model was applied to the ZINC v.7 database and afterwards experimentally validated by an enzymatic inhibition assay. One of the compounds selected by the model showed cruzain inhibition in the low micromolar range.