This study was conducted to reset the maximum residue limit (MRL) for didecyldimethylammonium chloride (DDAC) in broiler chickens. The disinfectant containing DDAC (10%, w/w) was diluted 160 times and evenly sprayed on the bodies of twenty-four broiler chickens at a rate of 15 mL per day per bird for 7 days. After the disinfectant treatment, tissue samples were collected from six broiler chickens at 0.25, 1, 3 and 5 days, respectively. Residual DDAC concentrations in poultry tissues were determined using LC-MS/MS. Correlation coefficient (0.99 >), the limits quantification (LOQ, 2.0~10.0 μg/kg), recoveries (86.9~118.6%), and coefficient of variations (<19.98%) were satisfied the validation criteria of Korean Ministry of Food and Drug Safety. In all tissues except for liver, DDAC was detected more than LOQ at 5 days after the disinfectant treatment. In liver tissues, DDAC was detected more than LOQ at 3 days after treatment. According to the European Medicines Agency’s guideline on determination of withdrawal periods, withdrawal period of DDAC in poultry tissues was established to 26 days. In conclusion, the developed analytical method is sensitive and reliable for detecting DDAC in poultry tissues. When DDAC disinfectant is sprayed on a poultry house in the presence of broiler chickens, it is necessary to keep the disinfectant from contacting the body of the livestock.