The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC (p)°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K (ITC), ΔH (ITC)). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal-buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.