Direct analysis in real-time mass spectrometry (DART-MS) works at atmospheric pressure without lengthy, labor-intensive sample preparation or chromatographic separation of analytes. Consequently, DART-MS reduces labor, most laboratory supply costs, and overall time of analysis. This study was designed to determine the usefulness of DART-MS for the rapid detection and semiquantitation of the β-agonists clenbuterol, ractopamine, salbutamol, and zilpaterol in cow, horse, and sheep urine. Samples were mixed with 10% sodium carbonate followed by extraction with ethyl acetate and applied to a 96-grid DART mesh prior to analyses. Total DART-MS analysis time was approximately 45 min per 96-grid plate. The coefficient of determination of standard curves for the compounds in cow, horse, and sheep urine ranged from 0.9930 to 0.9986, 0.9829 to 0.9990, and 0.9925 to 0.9997, respectively. Limits of quantitation (LOQs) for clenbuterol, ractopamine, salbutamol, and zilpaterol ranged from 4.0 to 239.7, 29.5 to 551.3, and 6.9 to 205.2 ng/mL in cow, horse, and sheep urine, respectively. The interday relative standard deviations (RSDs) of standards fortified in cow, horse, and sheep urine were 26.3 to 33.4, 23.9 to 41.1, and 30.7 to 37.0%, respectively. Moderate correlations were measured between DART-MS and LC-MS/MS data for incurred zilpaterol in horse (R2 = 0.73) and sheep urine (R2 = 0.59). The study demonstrates the utility of DART-MS for rapidly screening four commonly assayed β-agonists in urine matrices with very little sample preparation.