Clenbuterol (4-amino-α-[(tert-butylamino)methyl]-3,5-dichlorobenzylalcohol) is a β2-adrenergic agonist. The consumption of meat contaminated with clenbuterol can lead to increased heart rate, blood pressure, anxiety, palpitations and skeletal muscle tremors. Several analytical methods have been developed to identify and quantify clenbuterol in different biological matrices. In this report, we have developed a specific and sensitive analytical method for quantifying clenbuterol and performed an in-depth enantiomeric analysis in bovine urine. The method was evaluated in accordance with international guidelines, and we used an isotopically labeled analog as an internal standard. The extraction efficiency for clenbuterol in bovine urine was > 98%, the limit of detection was 0.05 ng/mL and the limit of quantification was 0.10 ng/mL. Our assay showed high specificity, no carryover was observed and the assay was linear in the range 0.10-8.0 ng/mL. Fifteen bovine urine samples were analyzed (containing clenbuterol), and an enantiomeric analysis was performed. The clenbuterol concentration range was 0.10-10.56 ng/mL across these samples. The levorotatory enantiomer was detected at greater concentrations than the dextrorotatory enantiomer, the ratio being 1.7 ± 0.6 (n = 15), and a statistical difference was observed (P < 0.05) using the Wilcoxon test.