The eggplant shoot and fruit borer (Leucinodes orbonalis) is a major pest of brinjal, causing significant yield losses due to increasing insecticide resistance. This study investigates the impact of sub-lethal doses (LD₃₀) of spinetoram, tetraniliprole and emamectin benzoate, combined with temperature variations (29 and 32 °C), on detoxifying enzyme activities in L. orbonalis larvae. Insecticide exposure significantly enhanced the activities of carboxylesterase (1.34- to 1.45-fold), glutathione S-transferase (1.73- to 1.86-fold) and cytochrome P450 (up to 2.6-fold) compared to the controls, with tetraniliprole inducing the highest responses. Thermal stress at 32 °C further amplified carboxylesterase and glutathione S-transferase activities compared to 29 °C, indicating enhanced detoxification under high temperature conditions. Larvae failed to survive at 22 and 37 °C, limiting enzymatic analysis to moderate temperatures. Among the plant extracts tested, Nyctanthes arbor-tristis (500 ppm) markedly increased cytochrome P450 activity, suggesting its potential as a synergist for resistance management. These findings highlight the role of detoxifying enzymes in mediating insecticide resistance and thermal resilience in L. orbonalis. Integrating biochemical markers into resistance monitoring and leveraging plant-derived compounds could improve sustainable pest management strategies to address challenges posed by resistance and climate variability.