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 tiamulin (TML) in broiler chickens as a part of PLS program introduction. Forty-eight healthy Ross broiler chickens were orally administered with TML at the concentration of 25 g/L (TML-1, n=24) and 50 g/L (TML-2, n=24) for 5 days through drinking water, respectively. After the drug treatment, tissue samples were collected from six broiler chickens at 1, 2, 3 and 5 days, respectively. According to the previously established analysis method, residual TML concentrations in poultry tissues were determined using LC-MS/MS. In TML-1, TML in all tissues was detected less than LOQ at 2 days after drug treatment. In TML-2, TML in liver and kidney was detected more than LOQ at 2 days after treatment. According to the European Medicines Agency’s guideline on determination of withdrawal periods, withdrawal periods of TML-1 and TML-2 in poultry tissues were established to 0 and 2 days, respectively. In conclusion, the estimated WT of TML in poultry tissues is shorter than the current WT recommendation of 5 days for TML in broiler chickens.