Eggplant fruit and shoot borer, Leucinodes orbonalis (Lepidoptera: Crambidae), is a destructive borer pest that infests eggplant crop from nursery stage to fruiting. This study aimed to evaluate the current insecticide resistance status of L. orbonalis collected from ten different locations of Tamil Nadu, India, using a fruit dip bioassay. Late second instar larvae were exposed to spinosad, spinetoram, thiacloprid and emamectin benzoate. Median lethal concentration (LC50) and resistance ratios (RR) were estimated for each population using a susceptible NBAIR population as a reference. The results showed that emamectin benzoate was highly toxic to L. orbonalis. All field populations exhibited a low to high level of resistance against spinosad (7.4 to 72.1 fold) and spinetoram (6.6 to 67.8 fold), while susceptibility to low levels of resistance was observed against thiacloprid (0.89 to 5.06 fold) and emamectin benzoate (1.48 to 11.29 fold). Additionally, detoxification enzyme assays were conducted for cytochrome P450 monooxygenases (CYP/MFO), glutathione S-transferases (GST) and carboxylesterases (CarE) revealing high specific activity in the Dharmapuri (DMP) population, correlating with resistance. Furthermore, quantitative real-time PCR analysis was conducted to assess detoxification gene expression in resistant (DMP) and susceptible populations after insecticide exposure, revealing significant overexpression of cyp306a1 and ce125 across the studied insecticides, particularly after 48 h, indicating their key role in resistance. These findings highlight the need for insecticide rotation with different modes of action to mitigate resistance development in L. orbonalis, providing valuable insights for sustainable pest management in Tamil Nadu and similar agricultural contexts worldwide.