The Egyptian cotton leafworm Spodoptera littoralis (Bosid.), one of the main pests in Egypt that causes damage to industrial, vegetable, and ornamental crops. The development of resistance and biochemical mechanism of S. littoralis to four insecticides (deltamethrin, spinetoram, pyridalyl and indoxacarb) were studied in the laboratory. Selection pressure in all experiments was carried out on 4 th instar larvae for six generations by the leaf dipping technique. At the end of selection, the results indicated that the resistance ratios (RR) were 43.79-, 29.69-, 13.09-and 33.39-fold for deltamethrin, spinetoram, pyridalyl and indoxacarb, respectively, compared with the parent strain. At the end of selection pressure, detoxifying enzyme assays revealed that the α-esterases activity levels for such insecticides were 5542. 64, 4024.44, 4223.64 and 3641.32, respectively compared with parent strain 447.89, whereas those of β-esterase activity were 4132.30, 2343.25, 2953.70 and 1294.04 for deltamethrin, spinetoram, pyridalyl and indoxacarb, respectively, higher than in the parent strain 400.61. In addition, there was a significant increase in levels of AChE, ACP, ALP and GSH activity at the end of selection with these insecticides in all selected generations. The results demonstrated that, treatment of S. littoralis with DMT, SPT, PYD and INC compared with the susceptible strain caused a significant decrease in the protein, carbohydrate and lipid contents.