Blue poison dart frogs (Dendrobates tinctorius azureus) are commonly found in zoological institutions and are becoming popular in the pet trade. Sedation or light anesthesia is required for safe and effective handling of this species. In this study, the effect of subcutaneous administration of alfaxalone at two different doses (20 and 30 mg/kg) was evaluated in juvenile blue poison dart frogs (N = 10) in a prospective, randomized, blinded, crossover study. Heart rate, pulmonic respiratory rate, various reflexes, and behavioral parameters were monitored after drug administration. Both protocols resulted in rapid induction of sedation (median [range]) with loss of righting reflex: 20 mg/kg, 10 (10–20 min) and 30 mg/ kg, 10 (10–10 min). The time (mean ± SD) to return to spontaneous movement (20 mg/kg, 39.6 ± 7.6 min and 30 mg/kg, 54.5 ± 9.9 min; P < 0.01) and full recovery (20 mg/kg, 42.6 ± 8 min and 30 mg/kg, 61.6 ± 9.1 min; P < 0.01) after alfaxalone administration were significantly longer in the 30-mg/kg group. The frogs in both groups continued to remain responsive to tactile stimulation and retained palpebral reflexes. Alfaxalone induced cardiopulmonary depression and periods of apnea at both doses. The subcutaneous administration of 20 and 30 mg/kg alfaxalone resulted in clinically effective sedation in blue poison dart frogs. Sedation levels achieved in this prospective study would be adequate for a variety of noninvasive clinical procedures.