A comparative NMR conformational analysis of three distinct tetrapeptide inhibitors of the Hepatitis C NS3 protease that differ at the 4-aryloxy-substituted P2 proline position was undertaken. Specifically, transferred nuclear Overhauser effect experiments in combination with restrained systematic conformational searches were used to characterize the orientation of the P2 aryl substituents of these inhibitors when bound to the NS3 protease. Differences between free and bound conformations were also investigated. Analysis of the results allowed the design of a new P2 aromatic substituent, which significantly increased the potency of our inhibitors. The bound conformation of a specific competitive inhibitor having this novel P2 substituent is also described, along with a model of this inhibitor bound to the NS3 protease. This NS3 protease/inhibitor complex model also supports a hypothetical stabilization role for the P2 residue of the substrates and/or inhibitors and further elucidates the subtle details of the binding of the P2 residue of substrate-based inhibitors.