Application of different antibodies and extraction methods results in a wide range of steroid metabolite concentrations obtained during noninvasive hormones monitoring. It makes regional comparisons of steroid concentration very difficult. We compared three methods for extraction of glucocorticoids metabolites in tiger feces to examine correct stress level in Bengal and Amur tigers in India and Russia respectively. The results obtained with three different extraction methods correlate with each other positively and significantly. The highest concentration of fecal glucocorticoids metabolites (FGCM) was found after the extraction of wet feces samples with 90% methanol. The level of FGCM was significantly higher in Bengal tigers in India than in Amur tigers in Russian Far East. The reasons might be related to tigers’ density or anthropogenic pressure.