Abstract: High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is a comprehensive analytical technology developed in recent years for structural identification, qualitative and quantitative detection of compound components. Acarbose is currently one of the most widely used drugs in the treatment of type Ⅱ diabetes, and it has important medical significance. In this experiment, based on the fragments mass spectrometric of acarbose and its analogs, HPLC-MS/MS method was established for rapid screening of acarbose and its analog-producing bacteria from dozens of fermentation broths with different sources. After the fermentation broth was purified by a cationic solid-phase extraction column, simple dilution and centrifugation, separation was performed with a Waters CORTECS UPLC C18+ chromatography column. Methanol-water was selected as the mobile phase with the speed of 0.2 mL/min under gradient conditions. At positive ion mode, twenty strains of fermentation broth from different sources were screened experimentally. They were monitored by multiple reaction monitoring, parent ion scanning mode to detect acarbose, the analogs, and unknown compounds with a core structure of C7N. By summarizing the characteristic product ion fragments of acarbose and its analogs, the cleavage pathways of acarbose and its analogs were summarized, and the common characteristic fragments of compounds with C7N units were obtained, which was m/z 304. The two isomers of acarbose and acarbose impurity A were determined by mass spectrum energy resolution curve. The analysis method can realize the screening of acarbose and its analogue-producing bacteria, and provides the breeding of acarbose and important experimental ideas of its analogue-producing bacteria.