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Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor that is activated by proteolytic\ud cleavage of its N-terminus. The unmasked N-terminal peptide then binds to the transmembrane bundle,\ud leading to activation of intracellular signaling pathways associated with inflammation and cancer.\ud Recently determined crystal structures have revealed binding sites of PAR2 antagonists, but the binding\ud mode of the peptide agonist remains unknown. In order to generate a model of PAR2 in complex with\ud peptide SLIGKV, corresponding to the trypsin-exposed tethered ligand, the orthosteric binding site was\ud probed by iterative combinations of receptor mutagenesis, agonist ligand modifications and data-driven\ud structural modeling. Flexible-receptor docking identified a conserved binding mode for agonists related\ud to the endogenous ligand that was consistent with the experimental data and allowed synthesis of a novel\ud peptide (1-benzyl-1H[1,2,3]triazole-4-yl-LIGKV) with higher functional potency than SLIGKV. The\ud final model may be used to understand the structural basis of PAR2 activation and in virtual screens to\ud identify novel PAR2 agonist and competitive antagonists. The combined experimental and\ud computational approach to characterize agonist binding to PAR2 can be extended to study the many\ud other G protein-coupled receptors that recognize peptides or proteins.