TocharacterizeandidentifymultipleconstituentsinDanhonginjection(DHI),afastultra-highperformanceliquidchromatographycoupledtoelectrosprayionizationquadrupoletime-of-flighttandemmassspectrometry(UHPLC-ESI-QTOF/MS)methodwasestablishedandvalidatedinthepresentstudy.Atotalof63compounds,including33phenolicacids,2C-glycosylquinochalcones,6flavonoidO-glycosides,4iridoidglycosides,6organicacids,5aminoacids,and3nucleosides,wereidentifiedortentativelycharacterized.Inconclusion,theUHPLC-ESI-QTOF/MSmethodisusefulandefficientforin-depthstructuralelucidationofchemicalcompoundsincomplexmatricesofherbalmedicinessuchasDHI.