Abstract The ethanolic extract of Vitex doniana stem bark (11.9 g) was subjected to a silica gel accelerated column chromatography and eluents fractions (150 ml aliquots) obtained were collected and monitored with thin layer chromatography (TLC). Fractions with similar Rf values from same solvents system were poled together. Phytochemical test of all the fractions were perform. Complete elution yielded 48 fractions (150 ml/fraction) which were pooled to 24 fractions and finally to eight (8) eight fractions and coded. Fraction Vd8-a (56 mg) has gave a single spot a white crystal compound coded V1 on checking with TLC and observed under Ultraviolet lamp. The Rf values was calculated to be 0.433 and melting point was found to be 241-243 °C uncorrected. The infra red spectrum of compound V1 shows prominent peaks that corresponds to OH str (3365 cm-1) and C=0 (1652 cm-1). The 1H NMR (400 MHZ) spectrum of compound V1 in DMSO-d6 displayed five singlet signals. It further showed a broad singlet at δ 5.58 integrated for 1 H is due to an olefinic H-atom adjacent to the carbonyl carbon atom. Three signals at δ 3.10` (d, J = 9.0 Hz, H-22), 3.59 (m, 1H, 2H-a) and 3.72 (m, 1H, 3H-e) each integrating for one proton is due to an oxymethine protons indicating that three oxymethine H-atoms were present in the compound. The 13C-NMR spectrum showed the presence of 27 Carbon atoms, suggesting that may be steroid skeleton and The DEPT-135 spectra showed the presence of five CH3, eight CH2, and seven CH groups, and seven quaternary C-atoms. The Molecular formula was established as C27H44O7 by HRES-MS positive ion mode m/z 481.3179. Based on the spectral analysis, the compound V1 is thus concluded to have ecdysteroid skeleton and conclusively conforms with 2β, 3β 14α, 20R, 22R, 25- hexahydroxy-5 β cholest-7-ene-6- one, commonly known as 20-hydroxyecdysone. This is the first time this compound was isolated from vitex doniana sweet.