From 2020, Korean Animal and Plant Quarantine Agency has reset the withdrawal time (WT) for veterinary drugs typically used in livestock in preparation for the introduction of positive list system (PLS) program in 2024. This study was conducted to reset the MRL for amprolium (APL) in broiler chickens as a part of PLS program introduction. Forty-eight healthy Ross broiler chickens were orally administered with APL at the concentration of 60 mg/L (APL-1, n=24) for 14 days and 240 mg/L (APL-2, n=24) for 7 days through drinking water, respectively. After the drug treatment, tissue samples were collected from six broiler chickens at 0, 1, 3 and 5 days, respectively. Residual APL concentrations in poultry tissues were determined using LC-MS/MS. Correlation coefficient (0.99 >), the limits quantification (LOQ, 0.3~5.0 μg/kg), recoveries (81.5~112.4%), and coefficient of variations (<15.5%) were satisfied the validation criteria of Korean Ministry of Food and Drug Safety. In APL-1, APL in all tissues except for kidney was detected less than LOQ at 3 days after drug treatment. In APL-2, APL in liver and kidney was detected more than LOQ at 5 days after treatment. According to the European Medicines Agency’s guideline on determination of withdrawal periods, withdrawal periods of APL-1 and APL-2 in poultry tissues were established to 3 and 2 days, respectively. In conclusion, the developed analytical method is sensitive and reliable for detecting APL in poultry tissues. The estimated WT of APL in poultry tissues is longer than the current WT recommendation of 2 days for APL in broiler chickens.