Determination method of nine diarrhea shellfish poisons by ultra performance liquid chromatography-mass/mass spectrometry (UPLC-MS/MS) in bivalve molluscs was established. Methods The samples were extracted by methanol and separated on a reversed phase ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm) column with acetonitrile-0.01% ammonium hydroxide as the mobile phase, with a flow rate at 0.2 ml/min and the column temperature of 30 ℃. The free toxins (AZA1,AZA2,AZA3,PTX2-b,OA,DTX1,DTX2,YTX and hYTX) in the samples were detected under positive and negative multiple reactions monitoring (MRM) mode through polarity switching between time segments. When any one or more toxins of OA, DTX1 or DTX2 were positive in the sample, the esterified OA, DTX1 and DTX2 were converted to the free form by alkaline hydrolysis and their hydrolyzed toxins were determined by UPLC-MS/MS and quantified by external standard method. Results The average recoveries ranged from 85.9% to 95.3% at three spiked concentrations with the relative standard deviation(RSD) of 2.76% to 4.18%. The detection limit(LOD)ranged from 4.4 to 9.9 μg/kg. The quantification limit(LOQ) ranged from 14.7 to 33.0 μg/kg. Conclusion The method was highly sensitive and simple,and was suitable for daily detection of diarrhea shellfish poisons in bivalve molluscs.