The objectives of current study were to isolate, purify and characterize bioactive tryptamine derivative from Crataeva nurvala, a well explored traditional Indian medicinal plant with historical evidence of efficacy in the treatment of neurological and antioxidant deficiency related disorders. In this study, chloroform fraction of ethanolic extract of Crataeva nurvala stem bark was analyzed using column chromatography (gradient elution technique) and thin layer chromatography (TLC) to isolate melatonin, a biogenic indoleamine compound. The structure of the isolated compound was determined by various spectrophotometric analysis like UV, IR, 13C and 1H NMR and mass spectroscopy. Mass spectrum of isolated compound showed a parent molecular ion (M+) peak at m/z 233.2 gm/mol correspond to the molecular formula C13H16N2O2. In the 1H NMR spectrum, singlet (s) at δH 3.79 corresponds to 3 H of OCH3, multiplet (m) at δH 6.7, 7.0, 7.7 denotes 4 Ar. Protons, singlet (s) at δH 1.9 assigned for 3 H of CH3CO, triplet (t) at δH 2.8 corresponds to 2 H of N-CH2, multiplet (m) at δH 3.27 denotes 2 H of indolyl CH2, singlet (s) at δH 10.41 assigned for 1 H of NH indole and singlet (s) at δH 8.01 corresponds to 1 H, NH of secondary amide. 13C NMR showed presence of total 13 carbon atom. Based on physical and spectral characteristics, the isolated compound was identified and reported for the first time as N-acetyl-5-methoxy tryptamine (Melatonin).