We present PIGEON-FEATHER, a method for calculating free energies of opening (∆Gop) at single- or near-single-amino acid resolution for protein ensembles of all sizes from hydrogen exchange/mass spectrometry (HX/MS) data. PIGEON-FEATHER disambiguates and reconstructs all experimentally measured isotopic mass envelopes using a Bayesian Monte Carlo sampling approach. We applied PIGEON-FEATHER to reveal how E. coli and human dihydrofolate reductase (ecDHFR, hDHFR) have evolved distinct conformational ensembles tuned to their catalytic cycles. We showed how two competitive inhibitors of ecDHFR arrest its ensemble in different ways, only one of which is compatible with vertebrate DHFR orthologs. Finally, we uncovered how the lac repressor (LacI) ensemble responds to binding its inducer molecule and operator DNA to regulate transcriptional activation.