This study was performed to screen α-glucosidase inhibitors from the actinomycete metabolites library by high throughput screening. Twelve strains of actinomycete were considered to be α-glucosidase inhibitors producing strains; then effective inhibitory strain PW409 was fermented and separated by bioactivity based HPLC, two fractions showing remarkable inhibitory activities; the two compounds were identified as 1-deoxynojirimycin (DNJ) and miglitol by mass spectrometry, comparing with authentic standards, and relevant literature. The quantification analysis of DNJ and miglitol by HPLC-MS/MS showed that the average concentrations of DNJ and miglitol in broth of strain PW409 were 11.2 and 95.8 mg/L, respectively. This is the first report about Streptomyces sp. products α-glucosidase inhibitor miglitol. The strain PW409 has potential application in biosynthesis and biotransformation of antidiabetes drug miglitol. The method can be utilized for new α-glucosidase inhibitors discovering and development from other inhibitory activity strains.Colegio de Farmacéuticos de la Provincia de Buenos Aires