Objective To develop and validate the ultra high-performance liquid chromatograph coupled with mass spectrometry (UPLC-MS/MS) method for simultaneous determination of 16 mycotoxins, including aflatoxins, ochratoxins, trichothecenes, fumonisins and so on. Methods Five grams of the sample was extracted with acetonitrile/water/formic acid (79∶20∶1, V/V). After centrifuged and diluted, the extraction was separated by Cortecs C₁₈(100 mm×3.0 mm,1.6 μm) analytical column under acetonitrile-0.1% formic acid gradient eluting, then was determined by UPLC-MS/MS. Dilution isotope internal calibration was used for qualification. Results The result showed good linearities(0.05-200 ng/ml) for 16 mycotoxins in the certain correlation ranges with the coefficients all above 0.99. The limits of quantification ranged from 0.1 to 50 μg/kg. The average recoveries of 16 mycotoxins at three spiked levels ranged in 72.67%-126.92% with relative standard deviations of 0.15%-16.83%. The accuracies were acceptable by detecting natural matrix quality control samples. Conclusion The method is rapid, simple, sensitive, reproducible and accurate for simultaneous determination of multiple mycotoxins in cereals and products in routine laboratories. It meets the the requirement of mycotoxins monitoring in relative food.