Methimazole(1-methyl-2mercaptoimidazole), an antithyroid medicine is widely used to treat hyperthyroidism by inhibiting the production of thyroid hormone. It has been also approved for the same purpose for cats in Korea, but there is information that is also used for livestock. Therefore, it is necessary to develop reliable and sensitive methimazole detection methods for food safety assessment. In this study, we developed Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify methimazole in various samples (beef, pork, chicken, egg and milk). The samples were extracted usi cartridge 500 mg. Through the CODEX guidelines (CAC/GL 71-2009), the developed method was validated to be able to detect and quantify methimazole with excellent linearity, precision, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were 0.004 mg/kg and 0.01 mg/kg, respectively. R-square value (coefficient of determination with calibration range) of the linear standard calibration curves was higher than 0.99. The recovery rates were ranged from 70% to 112%, with the coefficient of variability (CV) of less than 10%. This method can be used for the food safety assessment and monitoring for the imported and domestic livestock samples.