The endocrinological study by immunological methods allows elucidating mechanisms of response to environmental challenges and reproductive regulatory mechanisms in animals. However, it is often overlooked that immunological assays for the detection and quantification of steroid hormones require prior validation tests. In this study, the efficacy of a commercial enzyme immunoassays (EIA) was evaluated for the quantification of plasma testosterone (T) in males from a population of green iguanas (Iguana iguana) in semi-captivity. The enzyme immunoassay was validated for specificity, accuracy and precision. Testosterone concentrations obtained by EIA were compared to estimates obtained on the same samples by high-performance liquid chromatography (HPLC). The proposed protocol has shown linearity and parallelism, T recovery was found to be within 80-110% accuracy, and precision variation was <10%. The EIA method allowed the differentiation of the plasma T concentration of male iguanas during the reproductive season (29.7 ± 14.4 ng mL-1, n = 4) and outside the reproductive season (6.8 ± 2.0 ng mL-1, n = 4). The HPLC method has been able to detect concentrations of T only for those individuals during the reproductive season. The T concentrations obtained by the two methods were not statistically different (p > 0.05) indicating that the commercial EIA kit analyzed can be employed in the laboratory routine to quantify plasma T concentration and consequently differentiate the reproductive status of green iguana males.