Of the two independent processes by which enzymes lose activity with increasing temperature, irreversible thermal inactivation and rapid reversible equilibration with an inactive form, the latter is only describable by the Equilibrium Model. Any investigation of the effect of temperature upon enzymes, a mandatory step in rational enzyme engineering and study of enzyme temperature adaptation, thus requires determining the enzymes’ thermodynamic parameters as defined by the Equilibrium Model. The necessary data for this procedure can be collected by carrying out multiple isothermal enzyme assays at 3–5°C intervals over a suitable temperature range. If the collected data meet requirements for V max determination (i.e., if the enzyme kinetics are “ideal”), then the enzyme’s Equilibrium Model parameters (ΔH eq, T eq, ΔG ‡ cat, and ΔG ‡ inact) can be determined using a freely available iterative model-fitting software package designed for this purpose. Although “ideal” enzyme reactions are required for determination of all four Equilibrium Model parameters, ΔH eq, T eq, and ΔG ‡ cat can be determined from initial (zero-time) rates for most nonideal enzyme reactions, with substrate saturation being the only requirement.