Abstract: Spinosad is a fermentation product of Saccharopolyspora spinosa Martz and Yao with biological activity against a range of insects including Lepidoptera. The cotton leafworm, Spodoptera littoralis (Boisd.) is the most destructive phytophagous lepidopterous pests in Egypt for it causes various ravages not only for cotton plants, but also other field crops and vegetables. This study was conducted in order to evaluate the effect of spinosad which is a new mode of action of insecticides on the activity of esterases (acetylcholine and aliphatic esterase), non-specific esterases (α-and β-esterase), phosphatases (acid and alkaline phosphatase), transaminases (aspartate and alanine transaminase) and carbohydrate hydrolyzing enzymes (amylase and invertase) on 4th instar larvae of the laboratory and the field strains of the cotton leafworm S. littoralis (Boisd.). It can concluded that, the change of response to spinosad could be associated with the decrease in AchE activity, likewise the tested compound caused a disturbance in the activities of the other tested enzymes either with increase (Ali-E, β-E and amylase activities) or with decrease (ALT).