The cotton leafworm, Spodoptera littoralis (Boisd.) is a serious pest of cotton and other crops in Egypt. Bioassay was carried out to evaluate the toxicity of some biochemical insecticides (emamectin benzoate, spinosad, and Bacillus thuringiensis)against  2^nd larval instar of S. littoralis (Boisd).The LC₅₀ values determined for emamectin benzoate, spinosad, and Bacillus thuringiensis against S. littoralis larvae were 1.38, 114.09, and 54.19 ppm respectively. Based on the LC₅₀ values, emamectin benzoate was more toxic to S. littoralis than the other compounds. Biochemical studies were done to detect the effect of tested compounds on total carbohydrates, proteins, lipids, amylase, invertase, trehalase, and alpha and beta-esterase enzymes. The total results indicated that carbohydrates, total proteins, and lipids were decreased significantly after treatment with Bacillus thuringiensis than the other compounds. The tested insecticides significantly reduced the amylase, invertase, and trehalase activity. A significant increase in alpha and beta esterase was induced by all tested compounds.