The liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for determination of clenbuterol residues in liver tissue as a regulatory matrix for the control of clenbuterol abuse as an anabolic. To investigate the level of clenbuterol residues during the withdrawal period, male food-producing pigs were exposed to subchronic repeat oral administration of a clenbuterol growth-promoting dose for 28 days. The analytical procedure showed acceptable validation results for all liver spiked samples analyzed and proved to be useful as a quantification and confirmation method in supporting regulatory enforcement programs of clenbuterol misuse monitoring. The highest level of clenbuterol in the liver of treated animals was recorded on day 0 of treatment cessation (21.58 ± 14.29 ng/g), followed by 6.59 ± 3.11 ng/g on day 3, 0.83 ± 0.27 ng/g on day 7, and 0.44 ± 0.08 ng/g on day 14 of withdrawal. At the end of the study period (day 35), the concentration of clenbuterolwas below the method limit of detection ( < 0.1 ng/g).