This work aimed to study the effect of adding mint oil to Spinosad insecticide on the activity of some enzymes related to the toxicity of the compound against the cotton leaf worm, Spodoptera littoralis. The results showed that adding 0.3% mint oil to Spinosad solution increased its toxicity against S. littoralis 2^nd instar larvae, where its LC₅₀ value was reduced from 20.482 ppm to 8.068 ppm after adding mint oil. The biochemical studies in the treated larvae with Spinosad combined with 0.3% mint oil showed a significant decrease in the activity of the enzymes; AChE, β-esterase, and phenoloxidase, while acid and alkaline phosphatases activity significantly increased; compared to treatment with Spinosad only. This data confirms the effect of mint oil on the activity of these enzymes towards Spinosad, which may explain the increased toxicity of Spinosad when mixed with mint oil against S. littoralis.