Abstract: A rapid, accurate and simple HPLC-UV-ESI-MSⁿ method was developed for the separation and characterization of related impurities in puerarin bulk drug. The molecular structures and mechanism for the fragmentations of the related impurities were proposed by the MSⁿ results obtained, and confirmed by ESI-FTICR-MS which provided accurate molecular formulations at high mass resolution. The molecular structures of the four related impurities are identified as 3’-hydoxypuerarin, genistin-8-C-glucoside, neopuerarin B and neopuerarin A on the basis of fragmentation mechanism and reference data.