A method was developed for the determination of gliotoxin in food and feed by HPLC-MS/MS with pressurized liquid extraction (PLE) and auto-solid phase extraction (ASPE). The extracting solution were: acetonitrile/water (50/50;v/v) in plant samples; and acetonitrile/hexane (10/90;v/v) in animal samples. The elution solution was methanol-water (15/85;v/v) (ASPE). Qualitative analysis was performed with electrospray ionization in positive mode (ESI+) under multiple reaction monitoring (MRM) modes. The matrix-matched calibrations were used to quantify the residue concentrations. The calibration curves showed good linearity in the certain concentration for gliotoxin; with correlation coefficients (R2) more than 0.995 8. The average recoveries of gliotoxin were in the range of 81.8%-95.4% at three spiked concentration levels; and relative standard deviations were less than 13.2%. The limits of quantitation (LOQ) were in the range of 0.23-0.31 μg/kg. Conclusion: A simple and automated method has been successfully developed for rapid determination of gliotoxin in food and feed.