Abstract The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least 6 of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 milliseconds of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin’s binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R 2 =0.82). To test our ability to make blind predictions, we predicted blebbistatin’s binding affinity for an isoform (Myh7b) whose blebbistatin sensitivity was unknown. Encouragingly, we find good agreement between the predicted and measured IC50 (0.67 µM vs. 0.36 µM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets. Significance Drug development requires the discovery of compounds which specifically target one member of a protein family without triggering side effects that arise from interactions with other related proteins. Myosins are a family of motor proteins that are drug targets for heart diseases, cancer, and parasitic infections. Here, we investigate why the compound blebbistatin specifically inhibits some myosins more potently than others, even though its binding site is closed in all known experimental structures. We find that the blebbistatin binding pocket opens in molecular dynamics simulations of certain myosin motors, and that the probability of opening predicts how potently blebbistatin inhibits a particular motor. Our work suggests that differences in cryptic pocket formation can be exploited to develop specific therapeutics.