We describe a fluorescence-based assay for the analysis of xylanase activity using a novel fluorogenic substrate, 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside (DiFMUX(2)). Generation of fluorescent 6,8-difluoro-4-methylumbelliferone (DiFMU) from the substrate corresponded directly to xylanase activity. High-performance liquid chromatography analysis of the digestion products showed that xylanase hydrolyzed DiFMUX(2) directly to DiFMU and xylobiose. The assay provides the speed, sensitivity, and convenience required for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format and is suitable for the kinetic assay of xylanases from a variety of sources.