The fall armyworm, Spodoptera frugiperda is one of the most highly invasive and damaging agricultural pests. In Egypt, maize grains have enormous economic importance. Infestations of S. frugiperda in maize cause a significant reduction in maize yield. This research aimed to assess the potential impact of LC₅₀ values for chlorpyrifos, methomyl, and spinosad, set at 470, 105.5, and 2.5 ppm, respectively, on the biochemical and histopathological responses of S. frugiperda. The results indicated that the exposure of fall armyworm larvae to lethal concentrations of such insecticides resulted in a significant decrease in the total protein, carbohydrate, and lipids associated with a significant increase in α, β-esterase, and acetylcholinesterase. Moreover, a noted elevation in acid and alkaline phosphatases, Glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), phenoloxidase, and chitinase activities occurred. For the digestive enzymes, a significant decrease in amylase activity has occurred while the activities of invertase and trehalase have changed only with significant differences among spinosad and methomyl treatments. However, chlorpyrifos exhibited non-significant variations in the activities of invertase and trehalase. By the cross-section of the midgut larvae, distinct histological damage of the midgut was distinguished by cytoplasmic vacuolation, and necrosis with sloughing of epithelial lining from the basement membrane toward its lumen. In conclusion, all the treatments of insecticides, chlorpyrifos, methomyl, or spinosad, significantly affected the biochemical aspects and histopathology of S. frugiperda larvae.