The present study was designed to investigate the activities of the detoxifying enzymes acetylcholinesterase (AChE), non-specific esterases (α- and β-esterases), glutathione S-transferase (GST) and mixed-function oxidases (MFO) in spinetoram-treated Agrotis ipsilon (Hufnagel). For this purpose, fourth larval instars were exposed to three sub-lethal concentrations (LC₁₀, LC₂₀ and LC₅₀) of spinetoram for 24 h using the leaf dipping technique. The activities of detoxifying enzymes in the survivors after 2, 4 and 6 days of treatment were compared to non-treated larvae (control). AChE activity was significantly increased after 4 and 6 days of treatment with the LC₁₀. In contrast, this activity was significantly decreased after 2 days of treatment with the three sub-lethal concentrations applied, and after 4 and 6 days of treatment with the LC20 and LC₅₀. While both α- and β-esterase activities were significantly enhanced after 2 days of treatment with all the previous sub-lethal concentrations, with no constant pattern after 4 and 6 days of treatment. GST activity was significantly increased on the second day of treatment with the LC₁₀, and on the fourth day of treatment with the LC₁₀ and LC₅₀. Whereas the only significant decrease in GST activity was observed on the sixth day of treatment with the LC₁₀. No significant change was recorded on the second and sixth days of treatment with the LC₂₀ and L_C50, and on the fourth day of treatment with the LC₂₀. The activity of MFO was significantly enhanced up to the fourth day of treatment with all the concentrations investigated, whereas this activity was significantly decreased on the sixth day of treatment. Therefore, it appears that higher activities of detoxifying enzymes in A. ipsilon generally occurred in response to the intoxication by the lowest concentration of spinetoram, particularly after a relatively early time of treatment, and MFO may be considered the principal detoxifying enzymes.