Chromatographic separation of Catharanthus roseus yielded six fractions. Of the sex fractions, the alkaloid and butanol fractions were the most effective fraction against Spodoptera littoralis (Boisd.) larvae in their second instar after seven days of exposure using leaf dipping technique with LC50 value 234.58 and 481.09 ppm, respectively. Toxicological effect guided isolation and elucidation of the most toxic fraction and yielded three monoterpenoid indole alkaloids, vindoline (1), vindorosin (2) and ajmalicine (3) using extensive chromatographic and spectroscopic techniques. The effect of alkaloid and butanol fractions on biochemical aspects including transaminases enzymes (AST and ALT), acelylcolinesterase and chitinase for S. littoralis, 2 nd instar larvae were evaluated. A significant activation on AST and ALT activities were obtained after treatment with LC50 of alkaloid and butanol fractions also, both fractions might be used as AchE inhibitors and suggested an ability in biodegradation of chitin. The sublethal effect of both alkaloid and butanol fractions on the biological aspects of S. littoralis immature and adult stages were studied in details.