ITC is a quantitative technique that can determine the binding affinity (), reaction enthalpy (), and binding stoichiometry () of the interaction between two or more molecules in solution. This is achieved by measuring the enthalpies of a series of binding reactions caused by injections of a solution of one molecule to a reaction cell containing a solution of another molecule. The enthalpy values are plotted over the molar ratios resulting from the injections. From the plot, the molar reaction enthalpy , the affinity constant () and the stochiometry are determined by curve fitting. The reaction's Gibbs free energy change () and entropy change () can be determined using the relationship:
(where  is the gas constant and  is the absolute temperature).
For accurate measurements of binding affinity, the curve of the thermogram must be sigmoidal. The profile of the curve is determined by the c-value, which is calculated using the equation:
where  is the stoichiometry of the binding,  is the association constant and  is the concentration of the molecule in the cell. The c-value must fall between 1 and 1000, ideally between 10 and 100. In terms of binding affinity, it would be approximately from  ~ within the limit range.