The present study was carried out to induce rooting in single- and double-node cuttings of Momordica dioica Roxb. variety Indira Kankoda-2 (RMDSG-3) under controlled conditions and to investigate associated biochemical changes during root development. The cuttings were treated with six different concentrations of IBA (0, 1100, 1200, 1300, 1400, 1500 mg/l) at four soaking durations (5, 10, 15 and 20 second). Among five concentrations of IBA tested, 1500 mg/l IBA + 5 seconds soaking duration gave the highest percentage of rooting, number of roots, and root length in both single and double node stem cuttings. As a result, IBA 1500 mg/l was used further in this investigation for biochemical studies. The adventitious rooting was obtained in three distinct phases i.e., induction (0–12 days), initiation (12–14 days) and expression (14–18 days). IAA-oxidase activity of cuttings treated with IBA increased slightly as compared to control, this activity was found to decrease during induction and initiation phases and increase during the expression phase. The peroxidase activity of cuttings treated IBA-1500 mg/l increased up to initiation phase and decreased during the expression phase. Polyphenoloxidase enzyme activity/mg protein/min increased in both treated (0.9-1.75) and control (0.5-0.8) cuttings during induction and initiation phase but declined slowly during the expression phase. Total phenolic content was measured in mg/gm fresh weight, and it was found to be higher (6.55 mg/g fresh weight) in IBA- 1500 mg/l treated cuttings, particularly in initiation and expression phases, and it was also positively correlated with peroxidase activity. Phenolic compounds may be used as a rooting enhancer in spine gourd, and they can play a key role in inducing adventitious rooting. IBA is found efficient in spine gourd rooting, and by utilizing this protocol i.e., double node cuttings treated with 1500 ppm IBA for 5 seconds duration, spine gourd can be commercially propagating.