The aim of the study was to compare residue depletion of ractopamine HCl as a β-adrenergic agonist that promotes muscle growth of animals, from internal tissues on days after its repeat administration to animals. The experiment was carried out in 38 albino guinea pigs. Treated animals (n = 30) were orally administered ractopamine HCl in a dose of 3.5 mg/kg body mass per day for 7 consecutive days. On days 1, 10, 20 and 30 of drug discontinuation, animals were randomly sacrificed and the liver, kidney, lung, heart, muscle, spleen and fat samples were collected. In all matrices, ractopamine concentration was determined using validated enzyme-linked immunosorbent assay (ELISA) as a quantitative screening method. The highest ractopamine concentration was recorded on day 1 in the lungs (55.80 ± 15.62 μg/kg), followed by the kidney (21.85 ± 3.91 μg/kg), spleen (12.59 ± 1.95 μg/kg), fat (10.17 ± 5.02 μg/kg), heart (9.73 ± 0.22 μg/kg), liver (5.58 ± 2.09 μg/kg), and lowest in muscle (2.21 ± 1.02 μg/kg). Ractopamine residues were detected in the lungs in the period of 30 days after withdrawal in significantly higher concentrations in comparison to other investigated matrices, suggesting that depletion of ractopamine from the lungs occurs at a much slower rate than its depletion from other internal tissues.