North American vipers are commonly housed in zoological institutions or studied as free-ranging populations. Because of their venomous predatory and defensive mechanism, sedation or anesthesia is frequently employed to facilitate safe handling and medical procedures, especially of the head. A new formulation of alfaxalone with proprietary preservatives was recently approved and indexed for 28-d use post–vial puncture. Pharmacodynamic effects of alfaxalone in its prior formulation have been researched in nonvenomous species, but the optimal dose and route of administration in vipers have not been reported. In part one, 10 prairie rattlesnakes (Crotalus viridis) participated in a complete four-route crossover study evaluating 20 mg/kg alfaxalone administered intracoelomically (ICo), SC cranial to the heart, IM cranial to the heart, and IV in the ventral coccygeal vein. HR significantly decreased from baseline during IV (P= 0.024), IM (P= 0.024), and SC (P= 0.028) administration. Respiratory rate significantly decreased following alfaxalone delivered IV (P = 0.027). Time to first effects was significantly faster in IV compared with IM (P= 0.01), SC (P= 0.001), and ICo (P= 0.036). All IV and IM administrations resulted in deep sedation, but 70% of the IV and 10% of the IM sedation events resulted in apnea and required intermittent positive ventilation via endotracheal tube. Fifty percent of the ICo sedation events and 10% of the SC sedation events did not result in sedation. One successful SC sedation event resulted in apnea. In part two, echocardiograms were performed in the same rattlesnakes at baseline and at maximum effect of sedation with 20 mg/kg alfaxalone administered IM. Cardiac contractility and output were unaffected. Administration of alfaxalone at 20 mg/kg IM cranial to the heart should facilitate safe handling and minimally invasive procedures in prairie rattlesnakes and related species.