Using hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics we have identified mutation specific conformational changes, and provide a molecular framework for how they alter kinase activity.Many of the activating conformational changes mimicked previously described changes that occur upon membrane binding and allosteric inhibitor binding. We surveyed a diverse panel of PI3K inhibitors using HDX-MS and X-ray crystallography, and find that inhibitors that interact with the activation loop lead to allosteric conformational changes that partially mimic the R1021C mutation. Intriguingly, inhibitors that showed similar conformational changes to the activating mutant showed increased potency (~3 fold) for the mutant over wild type PI3K. Overall this work provides unique insight into the regulatory mechanisms that control PI3K kinase activity, and reveals a framework for the potential design of PI3K isoform and mutant selective inhibitors.