The dairy industry uses enzymes to make cheese, alter product flavor, and eliminate lactose. The activities of these enzymes have been measured in clear buffered solutions, but because of the limitations of spectrophotometric methods, enzyme activities have not been measured in opaque or colored dairy products where they are used. Isothermal titration calorimetry (ITC) can be used to determine reaction kinetics in opaque and colored solutions by measuring the heat rate from enzyme-catalyzed reactions as a function of time. This study used ITC to measure β-galactosidase activity in opaque solutions of milk, sweet whey, sweet whey permeate, acid whey, and acid whey permeate with two β-galactosidase (EC 3.2.1.23) isozymes derived from A. oryzae and K. lactis. The components of the dairy fluids alter the enzyme kinetics and reaction thermodynamics, and the reactions catalyzed by the two homologs differ as shown by differing thermodynamic profiles. The study demonstrates that ITC can be used to measure enzyme activity in opaque and colored dairy fluids and identify reactions by their thermodynamic properties. To ensure that ITCs are accurately recording heat data they must be calibrated regularly. However, potential problems have been identified with standard electrical calibration procedures; primarily being that the calibration is performed outside of the sample cell. This implies that any loss of heat from the theoretically adiabatic sample cell or loss of signal through led wires would be ignored by the electrical calibration. This research describes a new means for the chemical calibration of ITCs by performing acid-base titrations into the sample cell with KHP and TRIS base. This method for reaction was shown to be accurate to theoretical values across multiple temperatures and with different models of ITCs. Measurement errors due to diffusion of substrate are described along with means for limiting this factor. The method identified provides a procedure for maintaining the accuracy of ITCs by comparing their data to well-known thermodynamic values. It is anticipated that the simplicity and low-cost for running this calibration method will further standardize ITCs, help establish the ITC as a reliable method for measuring enzyme kinetics, and will make their maintenance simple enough for their use in quality assurance and industry settings.