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A study about the suitability of the chelation reaction of Ca²⁺with ethylenediaminetetraacetic acid (EDTA) as a validation standard for Isothermal Titration Calorimeter measurements has been performed exploring the common experimental variables (buffer, pH, ionic strength and temperature). Results obtained in a variety of experimental conditions have been amended according to the side reactions involved in the main process and to the experimental ionic strength and, finally, validated by contrast with the potentiometric reference values. It is demonstrated that the chelation reaction performed in acetate buffer 0.1 M and 25 °C shows accurate and precise results and it is robust enough to be adopted as a standard calibration process.