Endoxylanase (EC 3.2.1.8) substrate selectivity, i.e., its relative activity toward water-unextractable arabinoxylan (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, is important for its functionality in biotechnological processes such as bread-making and gluten starch separation. A screening method for rapidly determining said substrate selectivity was developed. Endoxylanase activity toward WU-AX was estimated by incubation of insoluble chromogenic substrate with a range of enzyme concentrations in microtiter plates, followed by colorimetric measurement of the dye released in the supernatant. A similar approach using soluble substrate and ethanol precipitation of unhydrolysed AX fragments was used to estimate enzyme activity toward WE-AX. A substrate selectivity factor was defined as the ratio of enzyme activity toward insoluble substrate over enzyme activity toward soluble substrate. A Bacillus subtilis and an Aspergillus aculeatus endoxylanase, known to have widely varying relative rates of hydrolysis of WU-AX and WE-AX, varied most in their substrate selectivity, while the endoxylanases of Aspergillus niger, Trichoderma longibrachiatum, and Trichoderma viride displayed intermediate such relative activities.