Abstract: α-Glucosidase inhibitors play an important role in the treatment of diabetes. This study established a high-resolution bioassay profiling platform for rapidly screening α-glucosidase inhibitors in natural product extracts. Five α-glucosidase inhibitors were identified from Malus hupehensis, namely, 3-hydroxyphloridzin, quercetin-3-O-β-D-glucopyranoside, phloridzin, avicularin and quercitrin. The establishment and successful application of this platform provides a powerful tool for the efficient discovery of anti-diabetic active ingredients in complex systems.