Background: The indiscriminating and intensive use of insecticides to control the cotton leafworm (CLW), Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), usually induces high levels of resistance. The insecticides are the principal method for controlling this pest because of its critical role in reducing insects when the economic threshold (ETL); therefore, the effectiveness of these insecticides should be maintained. So, the target of the present work was directed to focus on studying the change in the activities of some important enzymes as a result of sublethal treatment concentrations (viz. LC 25 values) of tested new insecticides (profenofos, cyfluthrin, emamectin benzoate, lufenuron, and spinetoram). The expected results could offer better understanding and more specific information about the resistance development in field populations of CLW because resistance is a significant challenge to pest control workers and these results may contribute to making the right decision at the right time. Materials and methods: Bioassays were performed on fourth instar larvae of S. littoralis field populations compared with the laboratory strain to assess the activity of the emamectin benzoate, lufenuron, and spinetoram by LC 25 and study the biochemical activities of some detoxification enzymes, like acetylcholinesterase (AChE), glutathione S-transferases (GST), alkaline phosphatase (ALP), and acid phosphatase (ACP) in fourth larval instar which was treated with LC 25 ; protein content is also determined.