Sex steroid hormone plays an integral part in mammalian reproduction. Quantitative estimation of these hormones from endangered carnivorous species like tigers is difficult by conventional method. The present study highlighted the estimation of ovarian steroids from faecal samples of captive royal Bengal tigresses. Faecal estrogen and progesterone concentration were measured at different phases of estrus period, during mating, at mid pregnancy and after parturition in five captive adult Royal Bengal tigresses (Panthera tigris tigris) of Nandankanan Zoological Park, Bhubaneswar. Faeces were collected within 18 hours after defecation in the morning. The sex steroid hormones were extracted from wet faeces and analysed by enzyme immunoassay (EIA) method. The overall value of estrogen and progesterone (ng/g) of tigresses were 1.45 ± 0.03 and 49.30 ± 23.78, respectively during breeding season. The overall mean (Mean ± S.E.M) values of estrogen (ng/g) were 1.49 ± 0.01, 1.52 ± 0.02 and 1.28 ± 0.04, in proestrus, estrus and post estrus period, respectively. Similarly, the faecal progesterone concentration (ng/g) recorded a value of 30.20 ± 3.36, 24.81 ± 3.52 and 72.08 ± 7.01 for corresponding estrus period.