Many chemical techniques exist for measuring the stoichiometry of ligand binding to a macromolecule; however, these techniques are often specific to certain ligands or require the presumption of specific binding models. Here, we further develop a previously reported, general, thermodynamic method for extracting the change in number of ligands bound to a macromolecule as that macromolecule undergoes a conformational transition driven by mechanical stretching, for example, by magnetic tweezers or optical trapping. We extend the theory of this method to consider systems with many ligands, experiments conducted in different thermodynamic ensembles (e.g., constant-force, constant-extension), and experiments in which the system is not at equilibrium. Further, we show that analysis of the same single-molecule mechanical manipulation data yields a measure of the differential free energy of stabilization due to ligand binding, that is, the free energy contribution by which ligand binding favors one conformation of the macromolecule over another. We interpret an existing data set measuring ion binding to RNA and DNA in terms of this free energy.