Abstract: A method for determination of 4 mycotoxins, including aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin M₁ (AFM₁), and zeralenone (ZEN) in grains by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with stable isotope labeling in vitro was proposed. The sample (200 mg) that passed through a 40 mesh (0.425 mm) sieve was taken and placed in a centrifuge tube. 0.5 mL of methanol was added, and the mixture was vortexed for 1 min and centrifuged for 3 min. The extraction process was repeated 3 times, and the supernatant was combined and blown to dryness by nitrogen. 170 μL of methanol was added to dissolve, and 20 μL of Girard P (GP) reagent (20 mmol·L^-1) and 10 μL of glacial acetic acid were added sequentially. The solution was vortexed for 10 s and shaken for 3 h at 50 ℃, and blown to dryness by nitrogen. 190 μL of methanol was added to dissolve, and 10 μL of GP-d₅ labeled mycotoxins standard (internal standard) solution (2.0 μmol·L^-1) was added. The 4 mycotoxins in the mixed solution were determined by LC-MS/MS. ODS-3 C₁₈ chromatographic column was used as the stationary phase and the mixed solution composed of 0.1% (volume fraction) formic acid solution and acetonitrile at different volume ratios was used as the mobile phase for gradient elution. Electrospray ion (ESI) source was used for mass spectrometry. The targets were detected by multiple reaction monitoring (MRM) mode in positive and negative ion scanning mode. As shown by the results, linear relationships between the ratios of the corresponding peak area to the internal standard peak area and mass concentrations of 4 mycotoxins were found in definite ranges, with detection limits (3S/N) in the range of 24-150 pmol·L^-1. Test for recovery was made by the standard addition method, giving results in the range of 79.2%-119%. Tests for precision of intra-day and inter-day were made, with RSDs (n=9) of determined values not more than 20%. This method was used for the analysis of 60 actual grain samples, and AFB₁, AFB₂, AFM₁, and ZEN were detected, with detection amounts in the range of 0.757-1.981 μg·kg^-1, 0.060-1.059 μg·kg^-1, 0.313-0.538 μg·kg^-1, and 0.969-4.348 μg·kg^-1, respectively.