|Abstract:||Alfaxalone, a neurogenic steroid with action at the γ-aminobutyric acid A receptor, is an injectable sedative frequently utilized in squamate medicine. A novel formulation of alfaxalone was recently approved by the Food and Drug Administration for 28-day use after breaching the vial due to the addition of proprietary preservatives. Previous research performed in squamates focuses on evaluating one route of administration at one or more doses and monitoring the effects of alfaxalone on that species (i.e. heart rate, respiratory rate, depth of sedation (Bertelsen 2011, Ferreira 2019, James 2018, Strahl-Heldreth 2019, Jurox 2020, Perrin 2017, Scheelings 2011). This thesis research examines the use of the new formulation of alfaxalone in two species: the bearded dragon and the prairie rattlesnake. Bearded dragons (Pogona vitticeps), a popular zoological companion species, frequently require sedation for procedures. Prairie rattlesnakes (Crotalus viridis) act as a model for that species and other Viperidae housed in zoological institutions or studied in free-ranging populations that require sedation to facilitate safe handling, examination, and minimally invasive procedures. A group of bearded dragons (n=10) and prairie rattlesnakes (n=10) were respectively sedated with alfaxalone at 15 mg/kg and 20 mg/kg based on a pilot study that identified the optimal dose for each species. A complete cross-over design evaluated four routes of administration: intracoelomic, subcutaneous, intramuscular, and intravenous. The optimal route of administration, defined as the route that provided consistent deep sedation with loss of righting reflex, maintenance of spontaneous respiration, and a clinically normal heart rate, was identified as intravenous (coccygeal vein) in bearded dragons and intramuscular (epaxials cranial to the heart) in prairie rattlesnakes. Echocardiography evaluated whether the cardiac function of either species was significantly affected by alfaxalone administered at the optimal dose and route of administration. End systolic volume was the only echocardiographic parameter that significantly increased in bearded dragons indicating a decrease in cardiac contractility secondary to sedation. Prairie rattlesnake echocardiographic values were not affected. Administration of alfaxalone at 15 mg/kg intravenously in the coccygeal vein of bearded dragons and 20 mg/kg intramuscularly in the cranial epaxials of prairie rattlesnakes should facilitate safe handling and immobilization for minimally invasive procedures in each species.