With the use of two fluorescence spectroscopic detectable substrates, L-phenylalanin-7-amido-4-methylcoumarin (L-PheAMC) and D-phenylalanin-7-amido-4-trifluoromethylcoumarin (D-PheAFC), it was possible to monitor a quasi-enantiomeric enzymatic reaction. The simultaneous hydrolysis of L-PheAMC and D-PheAFC was applied in aqueous media and via on-line 2D-fluorescence spectroscopy it was possible to draw conclusions about the enantioselectivity of the used enzymes alpha -chymotrypsin and esterase from porcine liver. The kinetic parameters from the single-hydrolysis of L-PheAMC and D-PheAFC were determined via a timescan run, the results showed, that the L-substrate was hydrolyzed 10(6) times faster than the D-substrate by alpha -chymotrypsin. In a simultaneous usage of both coumarin substrates with ar-chymotrypsin, the L-coumarin was favored by the enzyme, even when the L-substrate was used in a 10 times lower concentration than the D-substrate. With the unspecific esterase, the L-substrate was first hydrolyzed again when equal concentrations of both coumarins were used. But the reaction was influenced by the presence of the D-substrate. With a 10 times lower L-substrate concentration, the D-substrate was hydrolyzed by the esterase, but the reaction was again influenced by the presence of the enantiomeric partner.