We examined the role of the most important metabolic enzyme families in the detoxification of neurotoxic insecticides on adult males and females from susceptible populations of Cydia pomonella (L.), Grapholita molesta (Busck), and Lobesia botrana (Denis & Schiffermüller). The interaction between the enzyme families – carboxylesterases (EST), glutathione-S-transferases (GST), and polysubstrate monooxygenases (PSMO) – with the insecticides – chlorpyrifos, λ-cyhalothrin, and thiacloprid – was studied. Insect mortality arising from the insecticides, with the application of enzyme inhibitors – S,S,S-tributyl phosphorotrithioate (DEF), diethyl maleate (DEM), and piperonyl butoxide (PBO) – was first determined. The inhibitors' influence on EST, GST, and PSMO activity was quantified. EST and PSMO (the phase-I enzymatic activities) were involved in the insecticide detoxification in the three species for both sexes, highlighting the role of EST, whereas GST (phase-II enzymes) was involved only in G. molesta insecticide detoxification. L. botrana exhibited, in general, the highest level of enzymatic activity, with a significantly higher EST activity compared with the other species. It was the only species with differences in the response between sexes, with higher GST and PSMO activity in females than in males, which can be explained as the lower susceptibility of the females to the tested insecticides. A positive correlation between PSMO activity and the thiacloprid LD50s in the different species-sex groups was observed explaining the species-specific differences in susceptibility to the product reported in a previous study.