Neonicotinoid insecticides such as thiamethoxam (TMX), thiacloprid (TCD), imidacloprid (ICD) and acetamprid (AMD) are selective nicotinic acetylcholine receptors (nAChRs), and their use has been increasing exponentially. The work aimed to investigate the effects of these compounds on biochemical markers of the 4th larval instar of Spodoptera littoralis. TMX, TCD, ICD and AMD were individually mixed in artificial diet at field application rate. The fourth instar larvae were allowed to feed on contaminated or clean diets as untreated control for 1 and 24 hr. The biomarkers of total protein (TP), alanine aminotransferases (ALT) and aspartate aminotransferases (AST), alkaline phosphatase (ALP), antioxidants enzymes (superoxide dismutase (SOD) and glutathione-S-transferase (GST), acetylcholinesterase (AChE) and general esterases were determined in the whole larval homogenates. Results showed that ICD, significantly decreased TP, ALT, AST, ALP and AChE after 24 hr., of exposure. TCD, significantly decreased ALP and SOD (24 hr., of exposure) and GST (after 1 and 24 hr., of exposure). Unexpectedly, TCD induced AChE activity after 24 hr., of exposure. Colorimetric determination of esterases showed significant increase in activity after 1 and 24 hr., of exposure to ICD recording 268.6 and 282.1 nmol/min/mg protein compared with 92.3 and 100.8 nmol/min/mg protein of control, respectively. The obtained results were confirmed electrophoretically by native gel electrophoresis that showed high intensity bands in ICD lane. Also, significant increase in esterase activity was obtained in groups treated with TCD and AMD after 24 hr., of exposure. The current data revealed that the tested neonicotinoid insecticides have different adverse effects on biomarkers of S. littoralis that reflects different manners of insect response towards neonicotinoid with the important role of esterases in the tested insecticides detoxification.