Several investigations have shown that acid ceramidase inhibitors are potential antiproliferative and cytostatic drugs for cancer chemotherapy. The combinatorial chemistry approach for the discovery of acid ceramidase inhibitors requires the availability of a high-throughput enzyme assay. The synthesis of a novel fluorogenic ceramidase substrate, and its processing both in vitro and in cultured cells in a microtiter plate layout, are reported in this article. This coumarinic substrate was hydrolyzed in vitro (rat liver lysosomes) with Km and Vmax values of 113 microM and 3.6 pmol min-1 mg-1, respectively. Similarly, hydrolysis occurred in intact cultured cells that overexpressed acidic ceramidase. The assay was validated for the identification and characterization of acidic ceramidase inhibitors by using several alpha-ketoamide ceramide analogues, whose inhibitory activity had been previously described.