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A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for trace residue determination of bicozamycin (BZM) in livestock products and seafoods. BZM was extracted from a sample with acetonitrile-water (4 : 1), followed by a two-stage SPE enrichment and cleanup. The first stage involved a styrene-divinylbenzene copolymer cartridge (GL-Pak PLS-2), and the second stage involved a divinylbenzene-N-vinylpyrrolidone copolymer cartridge (Oasis HLB). The LC separation was performed on a C18 column using 0.01% formic acid-methanol (8 : 2) as the mobile phase and MS detection with negative ion electrospray ionization. The mean recoveries from swine muscle, liver, yellowtail, and milk fortified at the minimum residue limit (MRL) levels and 0.01 mu g/g were >70%, and the relative standard deviations (RSDs) were <20%. Limits of quantitation (LOQs) ranged from 0.002 to 0.005 mu g/g.