This study aimed to validate an enzyme-linked immunosorbent assay (ELISA) and create a reference interval for androstenedione from captive Texas tortoises (Gopherus berlandieri). Reference interval creation for androstenedione could allow comparison of an individual’s values to others of the species in an effort to evaluate excess or deficiency and/or provide the basis for diagnosis of reproductive health or capability. In other species, androstenedione is associated with puberty onset and specific reproductive diseases; however, values and function of this hormone remain poorly studied in reptiles. The 25 adult male tortoises sampled were maintained at Texas A&M University’s Winnie Carter Wildlife and Exotic Animal Center. Serum was assayed via a validated androstenedione ELISA. Male Texas tortoise serum androstenedione concentrations were found to be 5.40 ± 5.16 ng/ml (mean ± SD), with establishment of a reference interval of 0.57–18.58 ng/ml. Relatively increased androstenedione values were associated with increased carapace length, carapace height, and tortoise weight but not gular horn length of male Texas tortoises. This information may benefit the care, management, and reproduction of captive tortoises. Extrapolation should be considered for evaluation of other species until their own intervals are established. Further research should include determination of baseline values of androstenedione for both healthy and reproductively challenged female tortoises.