A method for determining the tranquilizers Acepromazine (ACP) and Atropine(ATP) in pig tissues (kidneys, livers, and porks) by using UPLC‐MS/MS has been developed. The liquid chromatographic separation and mass spectroscopy of the two substances have also been optimized. The justification data of the methods were: limit of detection (LOD) and limit of quantification (LOQ) 0.15 and 0.50 ng/mL for ATP, 0.16 and 0.5 ng/mL for ACP in all matrix samples. Calibration curves exhibited linearity in concentration varieties from LOQ to 12 ng/mL for ACP and LOQ to 20 ng/mL for ATP. Recovery, intra‐day precision, and inter‐day precision were determined at spiked analytes concentration of 2.0, 5.0, and 10 ng/mL for ACP and at analyte concentrations of 2.0, 6.0, 15.0 ng/mL for ATP. Recovery ranged from 98.26 to 119.43 % for ACP and 97.45 to 118.44 % for ATP. The effect of the matrix sample of the technique was found to be insignificant. The confirmed method has been applied to ascertain the content of two substances in the porks, kidneys, and livers of 16 pigs, injected Prozil Fort ‐ Veterinary Medicine (contained ACP and ATP) at different intervals. This work supplied a new method for scientists, which could be applied to determine the tranquilizers Acepromazine (ACP) and Atropine(ATP) in pig tissues.