Analytical method for mycotoxins were developed and validated. To eliminate the matrix effect, a solid phase extraction (SPE) was performed prior to LCMS analysis. Major mycotoxins were categized into the two groups based on their hydrophobicity by the SPE cartridge. Major mycotoxins such as nivalenol, deoxy-nivalenol and their derivatives were separated by reversed-phase mode with a PFP column followed by MRM based MS detection. This developed method was evaluated for the real sample such as barley and corn. The results were obtained with good calibration curve linearity as well as recovery and reproducibility conformed to the EU or other regulations.