Veterinary diagnostics often require the immobilization of amphibians; however, intramuscular administration of anesthetics may be harmful to amphibians given their thin epidermis. Alternatively, topical application or immersion may be less effective in desert-adapted species. Seven adult Colorado River toads (Incilius alvarius) were administered 20 mg/kg of alfaxalone either intramuscularly (IM, n=4) or by topical application (TOP, n=3). All animals belonged to a chronically Mycobacterium spp.-infected population and were to be culled following anesthesia achieved in this trial. Time to recumbency, heart and respiratory rate, and response to palpebral, corneal, nociception (toe pinch), and righting reflexes were evaluated and repeated every 5 minutes following induction. Anesthetic depth on a scale of 1 to 5 (no effect to excessively deep anesthesia) was recorded every 5 minutes. If no major effect was noted following 30 minutes, alfaxalone was repeated at 20 mg/kg. Recumbency was achieved only in the IM group (median 12 minutes, range 7–14 minutes). No loss of corneal, palpebral, nociception, or righting reflexes was achieved in the TOP group despite repeated alfaxalone administration, whereas these were absent only to a variable extent in the IM group. Anesthetic depth and heart rate differed significantly between groups throughout anesthesia as well as at time points of attempted blood sampling (p=0.03). Undesired side effects were skin irritation (n=1) in the TOP group and intermittent apnea (n=1) in the IM group. Intramuscular alfaxalone at 20 mg/kg may therefore be suitable for short, noninvasive procedures, whereas topical application cannot be advocated, and alternative routes are recommended.