Investigations were carried out to estimate the qualitative and quantitative variation of ecdysteroids in the larval hemolymph and pupae of Orthaga exvinacea. The analyses were done by using HPLC and FTIR. HPLC separations were carried out using Shimadzu system with a reverse phase column (C18) of 250×4.6 mm i.d. The HPLC separations were carried out in binary gradient for 20 min. Acetonitrile 15% was used as solvent A, trifluoro acetic acid (TFA 0.1%) as solvent B. The eluents were monitored at 242 nm using a UV–visible detector. The main component found in the hemolymph and pupae was 20-ecdysteriod. The titre of ecdysteroid in the pupae showed a higher value (1.23 µg/pupae) than the ecdysteroid in the hemolymph of 6th (0.70 µg/10 µL equivalent) and7th (0.60 µg/10 µL equivalent) instar larvae. Structural characterisation of ecdysteroids in the samples were obtained from FTIR spectra of the samples and standard. The major steroid component in all the samples were found to be 20E. There were 3 major absorption peaks, viz., for OH, C=O and C=C functional groups.