The failure of the traditional insecticides to control cotton leafworm Spodoptera littoralis led to severe losses in different crops as a result of resistance development.  This study aimed to evaluate the toxicity and biochemical activity of newly emerged insecticides, spinosad, emamectin benzoate and dinotefuran on 4^th instar larvae of Spodoptera littoralis. The results clearly illustrated that emamectin benzoate was more toxic than spinosad and dinotefuran according to LC₅₀ values 0.8, 201.7 and 3979.6 ppm, respectively. Additionally, the tested compounds resulted in a significant reduction in Ache activity by 50, 42 and 63.2%, respectively. On the contrary, there was significant increase of α – E by 5.3, 90.9 and 75.2%, β- E by 33.3, 29.7 and 44%, AST by 163.6, 181.8 and 63.6%, ALT by 263.2, 210.5 and 157.89%, ACP by 100, 200 and 200% and ALP activities by 250, 320 and 158%, respectively for the mentioned insecticides in comparing with untreated larvae.