Metabolic networks can provide insight into the biosynthesis pathways of natural products present in plant-derived medicines. Here, we primarily established a highly efficient and targeted method for the systematic screening of isoquinoline alkaloids from the Macleaya genus. A total of 392 potential alkaloids were detected, 204 of which were further identified according to their tandem mass spectrometry (MS/MS) spectra and the characteristic fragmentation patterns of references. A metabolic network of isoquinoline alkaloids from the Macleaya genus was then constructed based on the structural relationships, metabolic level differences, and the isotopically labeled [ring-(13)C(6)]-tyrosine feeding experiments. New biosynthesis pathways for well-known alkaloids (berberine, sanguinarine, and chelerythrine) in the Macleaya genus were proposed on the basis of the established metabolic network. This work marks the first comprehensive study of the metabolic network of isoquinoline alkaloids in the Macleaya genus and provides a template for constructing the metabolic networks of other plant-derived medicines. CI - Copyright (c) 2021 Elsevier Ltd. All rights reserved.