The sulcata or African spurred tortoise (Centrochelys sulcata) is a large tortoise species that is commonly kept in zoologic collections and as a pet. The objectives of this study were to establish reference intervals for selected biochemical analytes in clinically healthy captive sulcata tortoises and to evaluate the impact of blood sampling site and sex. Blood samples were collected from 60 tortoises from either the dorsal coccygeal (tail) vein or the subcarapacial venous plexus based on their body size. The packed cell volume and refractometric total solids (TS) were determined. The concentrations of selected plasma biochemical analytes were determined using the VetScan VS2 analyzer and included albumin, aspartate aminotransferase, bile acids, calcium, creatine kinase, globulins, glucose, potassium, sodium, phosphorous, total proteins (TP), and uric acid. The calcium-to-phosphorous ratio was calculated. Reference intervals were determined and evaluated for the potential effects of blood sampling site and sex. There were significant differences (P < 0.05) associated with the blood sampling site in TS, TP, phosphorus, and globulins, with higher values in samples from the tail versus the subcarapacial sampling site. No significant statistical differences were noted in the plasma biochemistry analytes between the sexes. Reading of the globulins by the analyzer failed in 36 of 60 of the samples and was largely associated with the subcarapacial plexus sampling site. The reference intervals defined by the VetScan analyzer in this study can be used for clinical medicine and conservation of this tortoise species. Sampling site was identified as a factor significantly affecting some blood analytes in this study; this factor should thus be taken into consideration when assessing sulcata tortoise health status and using this testing methodology.