In order to understand the essential structural features for chalcones as Trypaosoma cruzi inhibitors, three-dimensional pharmacophore hypothesis were built on the basis of a set of known Trypanosoma cruzi inhibitors selected from literature using PHASE program. Five point pharmacophore with three hydrogen bond acceptor (A), and two aromatic ring (R) as pharmacophoric features were developed. Amongst them the pharmacophore hypothesis AAARR11 yielded a statistically significant 3D-QSAR model with 0.9371 as R2 value and was considered to be the best pharmacophore hypothesis. The developed pharmacophore model was externally validated by predicting the activity of test set molecules. The squared predictive correlation coefficient of 0.870 was observed between experimental and predicted activity values of test set molecules. The geometry and features of pharmacophore were expected to be useful for the design of selective Trypanosoma cruzi inhibitors.