Inhibition of alpha-glucosidase is a therapeutic approach for diabetes. In this study, a method based on online liquid chromatography diode array detection tandem mass spectrometry and biochemical detection (LC-DAD-MS/MS-BCD) was developed to screen and identify alpha-glucosidase inhibitors from selected beverage extracts, including pu-erh tea (Camellia sinensis var. assamica), eagle tea (Litsea coreana Levi.), and radix glycyrrhizae (Glycyrrhiza uralensis Fisch.). As a result, two components, (-)-epigallocatechingallate (EGCG) and (-)-epicatechingallate (ECG), as potent alpha-glucosidase inhibitors, were found in pu-erh tea. The 1050 values of EGCG and ECG on alpha-glucosidase (EC 3.2.1.20, from Saccharomyces cerevisiae) were 175.1 and 246.9 mu M, respectively, and both were lower than that of acarbose (IC50 = 3553.0 mu M), a commercial alpha-glucosidase inhibitor. Kinetic studies revealed that both EGCG and ECG inhibited alpha-glucosidase activity in a noncompetitive manner. The study suggests that the developed LC-DAD-MS/MS-BCD system is a powerful tool for rapid screening and identification of alpha-glucosidase inhibitors in complex samples and that EGCG and ECG may be good candidates as alpha-glucosidase inhibitors.