A high-performance thin-layer chromatography (HPTLC) method was developed for quantification of α-amylase inhibitory activity and stigmasterol content in ant plant extracts. An improved HPTLC method for the determination of total free radical scavenging activity in samples using DPPH• is also reported. For quantification of α-amylase inhibitory activity, the developed HPTLC plate is dipped into an α-amylase solution, and the bioautogram is then incubated at 25 °C for 30 min under humid conditions. For visualization of enzyme inhibitory activity, the starch test with an iodine indicator solution is used. The blue zone observed comes from the starch-iodine complex formed from starch that was not hydrolyzed by the amylase due to enzyme inhibition by the compound(s) present in the sample. The area of the blue zones was used to compare and quantify relative α-amylase inhibitory activity in different extracts. Location of the blue zones (hRF) on the plate was used to detect compounds that are responsible for the α-amylase inhibitory activity. Relative α-amylase activity was not related to the antioxidant activity, but was highly correlated with the stigmasterol content in the sample extracts (R = 0.95). Therefore, plant sterols present in the extracts might be responsible for α-amylase inhibitory activities in the extracts. •The developed method for quantification of α-amylase inhibitory activity provides an efficient and effective tool that can be used to screen, detect and quantify α-amylase inhibitory activity in plant extracts.•The proposed protocol is easy to run, involves minimal sample preparation, with multiple samples able to be analyzed in parallel on the same chromatographic plate, in a short time.•There were significant differences in α-amylase inhibitory activity, stigmasterol content, and total free radical scavenging activity between methanol, ethanol, dichloromethane, and ethyl acetate ant plant extracts.