A method for the direct measurement of free Ca2+ and Mg2+ concentrations in the range of 1–100 mM by NMR spectroscopy is demonstrated. The method automatically corrects for the effect of ionic strength on the activity of the species in solution and works satisfactorily even when significant concentrations of competitive ions are present. The method requires only the measurement of the 1H chemical shifts of our reporter ligands, glycolate and sulfoacetate, and is easily implemented using NMR imaging techniques. As a proof of concept, we extract the thermodynamic binding constants and conformer distributions of analyte ligands using an in situ ion gradient. Existing approaches for the measurement of free Ca2+ or Mg2+ concentrations by NMR operate only at very low ion concentrations or else require careful recalibration for different sample conditions. By providing the free Ca2+ or Mg2+ concentrations, the proposed methodology significantly enhances the information obtainable via NMR investigations of ion-responsive systems.