Clenproperol is a β-adrenergic agonist that have a similar chemical structure and physicochemical property to clenbuterol, allowing it easy to be illegally used instead of clenbuterol in zootechnics and sports. This raises the need for monitoring of clenproperol residue in animal-derived foods entering the market destined for human consumption. Thus, a reliable and sensitive high performance liquid chromatography – tandem mass spectrometry (HPLC–MS/MS) method for the detection and identification of clenproperol residue in most commonly consumed animal-derived foods such as milk, yogurt, sausage, swine muscle, bovine muscle and goat muscle samples has been developed. The extraction and clean-up were carried out by employing an ammonium acetate solution and solid phase extraction with mixed-mode cation exchanger (MCX) cartridges. The developed method was fully optimized and successfully validated for all six matrices according to the National Standard of the People's Republic of China GB/T 27417–2017 and was demonstrated to be highly specific and sensitive. Quantitative analysis was performed by means of internal standard calibration using structurally similar clenbuterol-D9 as internal standard. The method presented low limits of detection (0.015–0.06 μg kg−1) and quantification (0.05–0.2 μg kg−1), and good specificity, linearity (R2 >0.999), precision and trueness, in terms of repeatability and in-laboratory reproducibility. The recoveries were between 76.1 % and 109.1 % in all matrices studied, with relative standard deviation (RSD) values in the range of 1.2–10.5 %. Furthermore, the method was successfully applied to the analysis of real samples, indicating the suitability of the proposed method for the determination of clenproperol. The developed method could be a valuable tool for control of illegal use of clenproperol in zootechnics.