Monitoring of physiological stress in wild and/or endangered animals living in their natural habitat can be generated via measuring faecal glucocorticoid metabolites (FGCMs) through enzyme immunoassay technique. However, a careful validation of each enzyme immunoassays (EIAs) and methodological issues such as samples preservation, and extraction procedures must be a significant concern and validated because it may influence FGCMs levels. In this present study, the aim was to carry out a biological validation to find reliable EIAs that could be used for non-invasive measurement of glucocorticoid levels in crested macaques. Moreover, we compared three different techniques of preservation: lyophilisation, oven drying and field extraction and three different solvents for extraction: 80% methanol, 90% pure ethanol and 90% commercial alcohol (isopropyl alcohol) to find the simple and practical techniques for sample processing. For hormone analysis, we colIected 189 faecal samples from wild crested macaques living in the Tangkoko-Batuangus Nature Reserve, North Sulawesi, Indonesia. The results show that biological validation can be used to validate measurement of faecal glucocorticoid which 3α,11β-dihydroxy-etiocholanolone was the most reliable EIAsfor this species. Different preservation and solvent extraction were not significantly influenced levels of FGCMs (p>0.05). In conclusion, Our study highlights the importance of biological validation of FGCMassays and presents practical techniques for the non-invasive monitoring of physiological stress in crested macaques.