Traditionally, testosterone is thought as a male hormone and estrogens as female hormones. However, estrogens and testosterone are produced by both sexes and appear to have important roles in both males and females. Measuring hormone concentrations in the wild without disturbing the animals is now feasible using non-invasive methods like fecal determination. These methods, however, need careful validation as the circulating hormone is usually degraded into several metabolites and it is necessary to verify that the antibody used in the immunoassay only recognized metabolites of the original hormone. Because the metabolites excreted in feces may be different for males and females, it is essential to validate immunoassays in both sexes. In this study, we determined whether the radioimmunoassays we previously used to measure fecal testosterone in male baboons and fecal estrogens in female baboons were suitable to measure these hormones in the opposite sex. We compared the hormone concentrations measured by radioimmunoassays to those determined by liquid chromatography combined with triple quadropole mass spectrometry (LC/MS/MS), a highly specific method that measures separately individual steroids. We also verified that fecal hormone concentrations reflected physiological levels. We found that estrogens and testosterone concentrations determined by radioimmunoassays were correlated, respectively, to 17-β-estradiol and testosterone determined by LC/MS/MS, for both sexes, suggesting that the metabolites measured by the two radioimmunoassays reflected the hormone of interest for both males and females. We also confirmed that the estrogens and testosterone concentrations determined by radioimmunoassays were physiologically relevant; estrogens and testosterone concentrations in adult males were higher than in immature males and testosterone concentrations in pregnant females were higher than in cycling and lactating females. These results led us to conclude that our radioimmunoassays can reliably measure testosterone and estrogens in both sexes, opening the field to investigations of the roles of testosterone in females and estrogens in males.