Non-invasive measurement hormones in feces using enzyme immunoassay (EIA) technique needs to be validated. This study was conducted to develop and validate an enzyme immunoassay kit for measuring the concentration of cortisol metabolites in feces of Toraya buffalo. An EIA kit of 11β-hydroxyetiocholanolone (11β-hydroxy-CM assay) was developed and validated analytically and biologically using feces of Toraya Buffalo for cortisol metabolite measurements. Analytical validation comprises the parallelism test, accuracy, precision, and assay sensitivity. Biological validation performed by comparing concentration cortisol metabolites in feces of Toraya buffalo before and after pa’silaga tedong, a fighting contest of buffalo during the death ceremony of rambu solo at Tana Toraja, South Sulawesi Province. Results showed that the curve of serial dilution of fecal samples was parallel with the 11β-hydroxy-CM standard curves. The accuracy and sensitivity of the 11β-hydroxy-CM assay were 96.21%±7.59 and 0.78pg/well, respectively. The precision based on coefficient variation (CV) of intraand inter-assay was < 10% and < 15%, respectively. Biological validation results showed that cortisol metabolites concentrations after pa’silaga tedong were increased 3-10 fold than before pa’silaga tedong. In conclusion, the 11β-hydroxy-CM assay is a reliable assay for measuring cortisol metabolites in feces of Toraya buffalo.