This study investigated chlorpheniramine maleate (CPM) residues in milk of intramuscularly dosed dairy cows and established the withdrawal time (WT) of CPM in milk. Sixteen healthy Holstein cows were injected with 10 (CPM-1) and 20 mL (CPM-2) of the drug containing 4 mg/mL of CPM, respectively. After administration of CPM, milk samples were collected from all cows at 12 hour intervals for 5 days. CPM residue concentrations in milk were determined using LC-MS/MS. The correlation coefficient of the calibration curve was 0.9956, and the limits of detection and quantification (LOQ) were 0.6 and 1.0 μg/kg, respectively. Recoveries ranged between 98.5-115.0%, and coefficient of variations were less than 10.96%. After treatment, CPM in CPM-1 and CPM-2 was detected below the LOQ in all milk samples at 12 hours. According to the European Medicines Agency’s guideline on determination of withdrawal periods for milk, withdrawal periods of both CPM-1 and CPM-2 in milk were established to 12 hours. In conclusion, the developed analytical method is sensitive and reliable for detection of CPM in milk, and the estimated WT of CPM in bovine milk will contribute to ensuring the safety of milk.