This paper describes a new spectrofluorimetric assay for continuously monitoring the enzymatic hydrolysis of medicinal esters. The procedure is based on the stoichiometric quantity of protons generated by the hydrolysis of the substrate, which produces changes in the fluorescence of a pH-sensitive dye. The pH indicator, 2', 7'-bis(car-boxyethyl)-5(6)-carboxyfluorescein, was selected due to its favourable pKa for studies under physiological conditions. Moreover, the presence of a domain in the spectra (< 442 nm) where fluorescence intensities are independent of pH allows measurements of wavelength ratios that cancel artifacts and lower sample-to-sample variability. The indicator did not affect the catalytic activity of purified hog liver carboxylesterase or human serum albumin. This assay is easy to perform and appears to be especially useful for studying enzymatic reactions with half-lives of the order of minutes or hours.