The aim of the study was to evaluate the adequacy of enzyme-linked immunosorbent assay (ELISA) in the post-exposure determination of the β-agonists clenbuterol and salbutamol in animal plasma and serum. Experimental guinea pigs (n = 20) were treated with two doses (0.25 and 2.5 mg/kg) of clenbuterol (n = 10) and salbutamol (n = 10) for seven days, whereas the control animal group (n = 10) was left untreated. Validation of the applied method yielded acceptable recovery (mean > 70%) and repeatability rates, showing ELISA to be applicable for the semi-quantitative determination of both analytes in both matrices, preferably in plasma. In both matrices, clenbuterol concentrations were proven to be significantly (14-fold) higher than those of salbutamol. Concentrations of both analytes were higher in plasma than in serum. The application of a 10-fold higher clenbuterol and salbutamol dose (2.5 mg/kg) resulted in concentrations 3- to 4-fold higher for clenbuterol and 2- to 3-fold higher for salbutamol, indicating a different release rate of these two β-agonists.