Eastern indigo snakes (Drymarchon couperi) have unique physiological features that are still not well understood. The objective of this study was to evaluate the effects of indoor vs. outdoor housing on plasma biochemistry analytes of eastern indigo snakes. Thirty-two captive eastern indigo snakes were divided into two groups: one group was housed indoors and the other group was housed outdoors in central Florida, USA. Packed cell volume and several plasma biochemistry analytes were evaluated: sodium, potassium, chloride, total magnesium, ionized magnesium, total calcium, ionized calcium, phosphorus, pH, glucose, uric acid, glutamate dehydrogenase, aspartate aminotransferase, creatinine kinase, total protein, protein gel electrophoresis, albumin (bromocresol method), cholesterol, calcidiol, and calcitriol. Significant differences were seen in plasma total calcium and calcidiol concentrations between indoor and outdoor female groups. Significant differences were also seen in packed cell volume and plasma total calcium and calcidiol concentrations between male and female eastern indigo snakes. All of the eastern indigo snakes in this study had markedly elevated plasma total calcium concentrations, similar to previous reports by others.