The current study investigated the role of four insecticides; Novaluron, Chromafenozidae, Thiocyclam and Chlorantraniliprole, in the biochemical activity of 2^nd and 5^th larval instars of cotton leafworm (Spodoptera littoralis). The total carbohydrates, proteins and lipids were determined in both larval instars under insecticide application. Also, the impact of tested insecticides on the activity of digestive enzymes,α- esterase, β- esterase, carboxyesterase, phenoloxidase, chitinase and acetylcholinesterase in S. littoralis larvae was studied. The results revealed that all tested insecticides decreased total protein and lipid content except chlorantraniliprole, which increased the total protein content. The tested insecticides increased the total carbohydrates and invertase activity except Thiocyclam. Applying Thiocyclam increased the trehalase and amylase enzyme content in treated larvae. Applying Chromafenozidae and Chlorantraniliprole reduced the activity of trehalase and amylase in the two larval instars. Chlorantraniliprole and Thiocyclam activated the activity of β- esterase and Carboxyesterase. In contrast, Novaluron and Chromafenozidae inhibited the content of β- esterase and Carboxyesterase enzymes. The activity of α- esterase was induced by applying Chromafenozidae and Chlorantraniliprole and inhibited by applying Novaluron and Thiocyclam on the two larval instars. Chlorantraniliprole activated Phenoloxidase and Chitinase enzymes in larvae, but Novaluron and Chromafenozidae decreased the activity of Chitinase. Acelylcolinesterase has been activated by applying Chromafenozidae and deactivated by Thiocyclam and Chlorantraniliprole. The obtained results lead to a better understanding of the tested insecticides' mode of action by studying the insicticides' influence on the biochemical activities of the treated larvae of S. littoralis.