The present article covers a simple approach to detect and subsequently identify in vivo metabolites of brodimoprim,using high performance liquid chromatography coupled to ion trap mass spectrometer(LC/ESI-MS),which is based on a data-dependent acquisition of isotope ions and result verified by full scan mass spectrum. The distinguished advantage of data-dependent scan is rapidness because it requires minimum sample preparation,and all the necessary data can be obtained in one chromatographic run. In addition,it is highly sensitive and selective,allowing detection of trace metabolites even in the presence of complex biomatrix. As a result,four phase-I(M1―M4) and four Phase-Ⅱ(M5―M8) metabolites of brodimoprim were identified in urine after the oral administration of brodimoprim to Wistar rats. Their chemical structures were proposed based on the interpretation of their CID fragmentation characterizations and the metabolic pathway was exhibited in this article. The present article covers a simple approach to detect and subsequently identify in vivo metabolites of brodimoprim, using high performance liquid chromatography coupled to ion trap mass spectrometer (LC / ESI-MS), which is based on a data-dependent acquisition of isotope ions and result distinguished by full scan mass spectrum. The distinguished advantage of data-dependent scan is rapidness because it requires minimum sample preparation, and all the necessary data can be obtained in one chromatographic run. In addition, it is highly sensitive and selective, allowing detection of trace metabolites even in the presence of complex biomatrix. As a result, four phase-I (M1-M4) and four Phase-II (M5-M8) metabolites of brodimoprim were identified in urine after the oral administration of brodimoprim to Wistar rats Their chemical structures were proposed based on the interpretation of their CID fragmentation characterizations and the metabolic pathway was exhibited in this article.