Introduction - Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of beta-glucosidase inhibitors but not for alpha-glucosidase. Nonetheless, alpha-glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti-viral infections. Objective - To develop a TLC bioautographic method to detect alpha- and beta-glucosidase inhibitors in plant extracts. Methodology - The enzymes alpha- and beta-D-glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of alpha-D-glucosidase, and 37 degrees C for 20 min in the case of beta-D-glucosidase. For detection of the active enzyme, solutions of 2-naphthyl-alpha-D-glucopyranoside or 2-naphthyl-beta-D-glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for alpha-D-glucosidase) or 1 :4 (for beta-D-glucosidase) and sprayed onto the plate to give a purple background colouration after 2-5 min. Results - Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited alpha-D-glucosidase and beta-D-glucosidase down to 0.1 mu g. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 mu g for alpha-glucosidase and 50 mu g for beta-glucosidase. Conclusion - The screening test was able to detect inhibition of alpha- and beta-glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright (C) 2009 John Wiley & Sons, Ltd.