Currently available tests for the diagnosis of inflammatory disease in reptiles are limited and poorly sensitive. However, a number of hematological and plasma biochemical analytes are validated in the diagnosis of inflammation in mammals. The objective of this study was to establish reference intervals for erythrocyte sedimentation rate, lactate, heat-precipitated fibrinogen, hematology, and plasma protein electrophoresis based on total protein by biuret method in 23 clinically healthy, captive gopher tortoises (Gopherus polyphemus) after successful rehabilitation and to determine differences by age, sex, and season. In order to investigate biological differences, samples were collected in April, July, and November. There were no sex differences in any measured analyte; however, there were significant differences by age and season. Immature animals (<2 kg) had significantly higher total protein, albumin : globulin ratio, pre-albumin, albumin, and α-1 globulin than adults. Tortoises sampled in the spring season had significantly higher total solids (refractometer) and lower eosinophils compared with animals sampled in the summer. Further investigation is required to determine the clinical utility of these analytes in the diagnosis of inflammation in this species.