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Thin-layer chromatography (TLC) coupled with bioassays has proven effective for detecting bioactive compounds in complex samples. In this study, a TLC enzyme-inhibition assay was developed for the detection of α-glucosidase inhibitors. The basic principle of the method involves the enzymatic hydrolysis of the substrate (2-naphthyl-α-d-glucopyranoside), resulting in the formation of β-naphthol that is subsequently reacted with Fast Blue B salt to produce a diazonium dye, resulting in a purple background. Assay development involved the utilization of enzyme gel entrapment, with either agar (over normal-phase plates) or poloxamer (over reversed-phase plates), and the optimization of key parameters including the substrate and enzyme concentrations, derivatization reagent concentrations, buffer type and pH, and plate type. The results showed good linearity within a range of 0.250–2.0 μg for the positive control, acarbose, with a coefficient of determination (r2) of 0.99. Detection and quantification limits were 0.060 μg and 0.199 μg, respectively. To address potential false positive results arising from secondary reactions with the reagents used, a nonenzymatic assay was conducted. In this control assay, the enzyme was replaced by the reaction product (β-naphthol). The developed TLC gel-overlay autographic method was able to detect the α-glucosidase inhibitor chlorogenic acid in a yerba mate extract. [ABSTRACT FROM AUTHOR]