The role of mixed function oxidases (MFO), hydrolytic cleavage enzymes and glutathione-mediated reactions in resistance of cotton leaf worm (CLW) Spodoptera littoralis (Boisd.) toward spinosad and abamectin were investigated using spinosad dipping resistant strain (SDRS), spinosad feeding resistant strain (SFRS) and abamectin dipping resistant strain (ADRS) and compared with the parent field strain (PS). Piperonyl butoxide (PB), MDPOC, triphenyl phosphate (TPP) and diethyl maleate (DEM) as mixed function oxidase, esterase and glutathione transferase inhibitors,respectively, were used in this investigation. The effect of cuticle permeability on the two mentioned insecticides was also studied by injecting the fifth instar larvae of S. littoralis with the two insecticides and treating them topically. Results emphasized that no role of cuticle penetration was found as resistance mechanism in the two spinosad resistant strains (SDRS and SFRS). While, cuticle permeability of ADRS was considered a responsible factor for the resistance of cotton leaf worm to abamectin. Values of synergistic ratio (SR) of spinosad in SDRS were 0.89, 1.11, 0.80 and 1.78 with PB, MDPOC, TPP and DEM, respectively. While SR values of spinosad in SFRS with the same corresponding synergists were 1.70, 1.96, 1.22 and 2.05, respectively. No significant differences were found between spinosad toxicity alone or with any of tested synergist in both strains SDRS and SFRS. These results suggest that metabolic detoxification enzymes play very limited role in the resistance mechanism(s) to spinosad in cotton leafworm (CLW). Values of SR in ADRS with the same corresponding synergists were 3.33, 2.29, 3.08 and 3.33, respectively. While SR values in the parent field strain were 1.08, 1.00, 0.97 and 1.03, respectively, with the same corresponding synergists. These results indicated high activity of detoxifying enzymes in ADRS compared with the parent field strain.