Abstract Isothermal titration calorimetry (ITC) is a powerful technique for probing biomolecular interactions. However, accurate determination of binding parameters—such as enthalpy and free energy—as well as associated uncertainties can be hindered by noise and concentration variability. Notably, the mathematical ambiguity surrounding analyte concentrations in standard binding models intrinsically limits the precision with which binding parameters, particularly binding enthalpies, can be determined. Here, we pres…