Chemical modulators of mechanosensitive Piezo channels would help better understand physiology and could treat certain diseases. A promising small molecule to expand the currently limited Piezo channel pharmacome is Yoda1, a Piezo1-selective activator identified from blind high-throughput screening. We previously uncovered an allosteric Yoda1 binding pocket in Piezo1, sandwiched between two hydrophobic transmembrane domains called Repeats A and B. Here, we show that crosslinking these Repeats with disulfide bridges inhibits the effects of Yoda1, supporting a mechanism in which Yoda1 acts like a wedge by separating these two domains from each other. Using open and shut Piezo1 structural models and rigorous in silico ligand binding free energy calculations, we next show that Yoda1 interacts with this binding region with a higher affinity for the open state, as expected for an allosteric activator. Our free energy calculations further recapitulate structure activity relationships established for seven Yoda1 analogs. Among 155 purchasable compounds virtually selected against a 3D pharmacophore map of the Yoda1 binding site in the open state, several act as bona fide Piezo1 activators with chemical scaffolds distinct from Yoda1 or other known Piezo1 modulators. This work provides a structural and thermodynamic framework for the activation of Piezo1 by Yoda1 and demonstrates the possibility to computationally exploit this pharmacological region to rationally design novel molecules with potential clinical value.