Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to in vivo conditions. The specificity constant, k(S) , is the ratio between k(cat) (the catalytic constant) and K(m) (Michaelis-Menten constant), and expresses the efficiency of an enzyme as a catalyst. This parameter is usually determined for purified enzymes, and in this work, we propose a classroom experiment for its determination in situ, in permeabilized yeast cells, based on a method of external enzyme addition, which was previously reported. Under these conditions, which resemble the in vivo state, enzyme concentrations and protein interactions are preserved. The students are presented with a novel approach in enzymology, based on the titration methods that allow the measurement of the enzyme amount, and thus the k(cat) and k(S) . The method will also be used to investigate the effect of exposure to oxidative stress conditions on yeast glyoxalase I.