Abstract: A scopoletin-based fluorimetric assay for the measurement of hydrogen peroxide formed by aldehyde oxidase was developed using sodium azide as an inhibitor of scavenging enzymes. The assay involves the coupled oxidation of scopoletin by hydrogen peroxide, catalysed by horseradish peroxidase and subsequent measurement of the change in scopoletin fluorescence. Hydrogen peroxide concentrations were measured against scopoletin fluorescence using two different scopoletin standards ranging from 0.1-2 and 4-40 μM. The overall linear correlation coefficient (r) for hydrogen peroxide concentrations ranging from 0.1-40 μM against fluorescence response is 0.998 (n = 3). The initial rates of hydrogen peroxide production by aldehyde oxidase were similar for both nitrogen heterocycles and aldehyde substrates. Initial rates of hydrogen peroxide formation during the oxidation of 100 μM substrates, indole-3-aldehyde or 2-pyrimidinone by aldehyde oxidase in the presence of 1 mM sodium azide were found to be 86-89% of substrate oxidation. All specific aldehyde oxidase inhibitors such as chlorpromazine (p<0.001), menadione (p<0.05) and β-estradiol (p<0.001), caused highly significant inhibition of hydrogen peroxide production during the oxidation of phthalazine, indole-3-aldehyde, 2-pyrimidinone and phenanthridine by guinea pig liver aldehyde oxidase. The kinetic constants, Km and Vmax, for hydrogen peroxide production during phthalazine and indole-3-aldehyde oxidation by guinea pig liver aldehyde oxidase have been determined for the first time using this method.