Despite considerable efforts, elucidating the allostery of large macromolecular assemblies at a molecular level in solution remains technically challenging due to its structural complexity. Here we have employed an approach combining amide backbone hydrogen/deuterium exchange coupled with mass spectrometry, fluorescence spectroscopy, and molecular simulations to characterize allosteric patterns of chaperonin GroEL, an similar to 800 kDa tetradecamer from E. coli. Using available crystal structures of GroEL, we quantitatively map out GroEL allosteric changes in solution by resolving exchange behaviors of 133 overlapping proteolytic peptides, with more than 95% sequence coverage. This comprehensive analysis gives a refined resolution down to five residues to pilot the GroEL allosteric determinants, of which the localized dynamics is monitored by tryptophan-mutated GroEL. Furthermore, the GroEL conformational transition is evaluated by molecular dynamics simulations with an atomic-interaction-based coarse-grained model. Collectively, we provide a practical methodology to analyze GroEL allostery in solution.