During the rational drug design, it is important to accurately determine the protein-ligand binding constant (the affinity or the Gibbs energy change upon binding), the enthalpy, and the entropy change upon binding. These three thermodynamic parameters of the association reaction are often determined by the isothermal titration calorimetry (ITC). Here, the repeatability of the measurement of the Gibbs energy and enthalpy of acetazolamide interaction with the recombinant human carbonic anhydrase II (CA II) was evaluated by four isothermal titration calorimetry (ITC) instrument models: PEAQ-ITC, VP-ITC, and iTC200 (manufactured by Malvern Instruments) and Affinity ITC (by TA Instruments). The protein-ligand binding reaction was repeated under identical conditions to obtain the statistical standard deviation of the measurements performed at 25 °C at various protein concentration. The enthalpy and Gibbs energy mean values plus minus the standard deviation of acetazolamide binding to CA II at 10 µM and 25 °C were equal to ΔH= −51.1 ± 3.1 kJ/mol and ∆G= −45.2 ± 1.3 kJ/mol as determined by averaging the results of all four instruments and applying the NITPIC-SEDPHAT software analysis that usually reduced the standard deviation of the enthalpy.