In 2019, researchers first discovered the highly invasive nocturnal pest Spodoptera frugiperda in Upper Egypt. This work investigated the sensitivity of S. frugiperda to six commercial pesticides in Egypt compared to Spodoptera littoralis and detected some biochemical changes (Detoxifying enzyme activities and protein patterns) in treated larvae of both insects. The results indicated that S. frugiperda was more sensitive to Spinosad (LC₅₀ = 6.23 ppm) and Abamectin (LC₅₀ = 27.75 ppm) than S. littoralis (LC₅₀ = 22 and 30 ppm, respectively). However, S. frugiperda was more resistant to Tebufenozid (LC₅₀ = 283.06 ppm) and Thiamethoxam (LC₅₀ = 269.4 ppm) than S. littoralis (LC₅₀ = 64.2 and 41.97 ppm, respectively). In addition, in comparison with S. littoralis, S. frugiperda was less sensitive to Alpha-Cypermethrin (LC₅₀ = 53.05 ppm) and Indoxacarb (LC₅₀ = 10.85 ppm), whereas S. littoralis was more sensitive to both pesticides (LC₅₀ = 5.02 and 4.02 ppm, respectively). The data of detoxifying enzyme (AST, ALT, ACP, ALP, and GST) activities indicated that the evaluated pesticides led to different extents of inhibition or activation of estimated detoxifying enzymes in both insect species. The different degrees in the detoxifying enzyme activities may be the causes of the variations of the insect sensitivity or resistance to the tested pesticides. The SDS-PAGE analysis indicated that there were variations in the protein patterns among S. littoralis and S. frugiperda when treated by tested pesticides. Thus, this study recommended that Spinosad, Indoxacarb, and Abamectin could be used to control S. frugiperda.