The development of resistance and biochemical mechanism of the cotton leafworm, Spodoptera littoralis (Boisd.), to five insecticides (chlorpyrifos, profenofos, cypermethrin, spinosad and flufenoxuron) were studied in the laboratory. The parent strain of S. littoralis was collected from El-Fayom Governorate at the cotton season 2005 and reared under laboratory conditions for seven generations, then subdivided into five sub-strains, three of them were selected by chlorpyrifos, profenofos and cypermethrin for fourteen generations and two were selected by spinosad and flufenoxuron for twelfth generations at LC₃₀ level. Selection pressure in all experiments was carried out on 4^th instar larvae by the leaf dipping technique. At the end of selection, the results indicated that the resistance ratios (RR) were 16.30-, 40.23-, 1070.42-, 145.14- and 8.24-fold for chlorpyrifos, profenofos, cypermethrin, spinosad and flufenoxuron, respectively, compared with the parent strain. At the end of selection pressure, detoxifying enzyme assays revealed that the α-esterase activity levels for such insecticides were 4.64, 4.67, 3.10, 3.40 and 1.24 times, respectively, higher than in the parent strain whereas those of β-esterase activity were 4.16, 4.07, 3.11 and 3.64 times for chlorpyrifos, profenofos, cypermethrin and spinosad, respectively, higher than in the parent strain, on the contrast the β-esterase activity for flufenoxuron was 0.96 time lower than in the parent strain. In addition, the levels of glutathione S-transferase (GST) activity at the end of selection with these insecticides were 6.62, 6.43, 2.11, 4.69 and 1.32 times, respectively, higher than in the parent strain. The results showed a correlation between the activity of both non-specific esterases (α- and β-esterases) and GST and resistance level to the tested insecticides. The results, also, indicated that the broad spectrum of insecticide observed in the field populations was due to multiple resistance mechanisms, including their increased detoxification. Finally, the rapid assessment of esterases and GST activities may be useful for monitoring resistance to these insecticides in S. littoralis. Therefore, differential levels of such enzymes could likely be used to detect the development of resistance during the early stages of insecticide resistance in the field.