A simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k−1), acylation (k2), and deacylation (k3) in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis–Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+ binding and site-directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.