The use of beta-blockers to enhance performance in some sports is forbidden. Based on this regulation, there is a demand for dynamic analytical procedures for analyzing these compounds quickly and without manual sample preparation. Therefore, the use of a molecularly imprinted polymer (MIP) in a multidimensional liquid chromatographic system coupled to a mass spectrometer provides a good alternative for improving the selectivity and practicality of the beta-blocker analyses, as described in this paper. A water-compatible MIP for oxprenolol was synthesized by the precipitation method, using methacrylic acid as a functional monomer and 2-hydroxyethyl methacrylate and glycerol dimethacrylate as hydrophilic monomers. A column filled with MIP was coupled to an LC-MS/MS instrument under the multidimensional configuration, with 10.0 mmol L(-1) ammonium formate buffer (pH 5.0) as the loading and reconditioning mobile phase and a 0.01% formic acid aqueous solution-methanol (30 : 70 v : v) as the elution mobile phase. The system was used for on-line extraction and quantization of oxprenolol (from 1.0 to 75.0 μg L(-1)), atenolol, propranolol, nadolol, pindolol, labetalol and metoprolol (all from 3.0 to 50 μg L(-1)) simultaneously, from urine samples. The correlation coefficient was higher than 0.99 for all the analytes. Suitable precision and accuracy were obtained.