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Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding it is necessary to define, at a microscopic level, the thermodynamic consequences of binding of each ligand to its energetically coupled site(s). However, extracting these microscopic constants is difficult for macromolecules with more than two binding constants. This goal is complicated because the observable (e.g., NMR chemical shift changes, fluorescence, enthalpy) can be altered by allostery, thereby distorting its proportionality to populations of states. Because it measures mass, native mass spectrometry (MS) can directly quantify the populations of homo-oligomeric protein species with different numbers of bound ligands, provided the populations are proportional to ion counts and that MS-compatible electrolytes do not alter the overall thermodynamics. These measurements can help decipher allosteric mechanisms by providing unparalleled access to the statistical thermodynamic partition function. We used native MS (nMS) to study the cooperative binding of tryptophan (Trp) to Bacillus stearothermophilus trp RNA-binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. Mass spectrometrycompatible solutions did not significantly perturb protein structure and thermodynamics as assessed by ITC and NMR spectroscopy. Populations of Trpn-TRAP11 states were quantified as a function of Trp concentration by native mass spectrometry. Population distributions cannot be explained by a non-cooperative binding model but are well described by a nearest neighbor cooperative model. Non-linear least-squares fitting of the populations to a mechanistic model yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites that result in homotropic cooperativity in Trp binding to TRAP.