A systematic stay of the cross-reactivities of β-adrenergic agonists and antagonists and their metabolites with an ELISA test designed to detect terbutaline and other β2-agonists has been performed in order to evaluate the applicability of this immunological test as screening method in routine human sports drug testing. Concentration response curves were calculated. IC50 values from 1.7 to 21.5 ng/mL were obtained for β2-agonists, and values from 3.1 to 520.8 ng/mL were obtained for most of the β-blockers. The highest cross-reactivity was mainly depending on the amino substituent of the molecules although the structure of the aromatic ring was also important. Minor changes in the aromatic ring, such as 4-hydroxylation for propranolol, did not largely influence the IC50 value. Distribution of control activities (percentage of optical density as compared with a blank sample) of urines obtained in controlled excretion studies (70 samples) and urines collected after competition in sports in which β-blockers are recommended to be tested (147 samples) showed a very small overlapping between the subpopulations of positive and negative samples. Cutoff values of 45 or 60% control activity were proposed for routine analysis. Sensitivity and specificity values of 92.8 and 98.8%, respectively (cutoff 45%), or 98.2 and 95.0%, respectively (cutoff 60%), were obtained. Because the known prevalence of positive results of β-blockers and β2-agonists in the target population was very low, the predicted percentage of presumptive positive cases that would need further confirmation by gas chromatography-mass spectrometry was also low, accounting for a 1.48 and 5.28% (for the cutoffs of 45 and 60%, respectively) of the total number of samples to be screened by the presented ELISA test. Thus, in terms of time and cost savings, the ELISA test is a powerful tool for the purpose of screening for β-adrenergic drugs in human urine.