Simple SummaryDrosophila suzukiiis a major destructive insect pest with a pandemic distribution. The lack of effective green control measures for this pest has prompted the search for new approaches, among which are peptides from animal venom. In this study, the biological activity of two underexplored spider venom peptides (J-atracotoxin-Hv1c and µ-theraphotoxin-Hhn2b) was assessed against adultD. suzukiiflies, as well as the biological response of flies to these peptides through detoxification mechanisms. Results demonstrate that µ-theraphotoxin-Hhn2b enhanced fly longevity. Gene expression analysis suggests that detoxification and stress-related mechanisms are triggered inD. suzukiiflies in response to treatment with these peptides. Our results highlight the potential of venom peptides to controlD. suzukii, underscoring the issue of how to ultimately devise improved target-specific formulations.AbstractThe spotted-wing drosophila (Drosophila suzukii) is a polyphagous pest that causes severe damage and economic losses to soft-skinned fruit production. Current control methods are dominated by inefficient cultural practices and broad-spectrum insecticides that, in addition to having toxic effects on non-target organisms, are becoming less effective due to acquired resistance. The increasing awareness of the real impact of insecticides on health and the environment has promoted the exploration of new insecticidal compounds, addressing novel molecular targets. This study explores the efficacy of two orally delivered spider venom peptides (SVPs), J-atracotoxin-Hv1c (Hv1c) and µ-theraphotoxin-Hhn2b (TRTX), to manageD. suzukii, through survival assays and the evaluation of gene expression associated with detoxification pathways. Treatment with TRTX at 111.5 µM for 48 h enhanced fly longevity compared with the control group. Gene expression analysis suggests that detoxification and stress-related mechanisms, such as expression of P450 proteins and apoptotic stimuli signaling, are triggered inD. suzukiiflies in response to these treatments. Our results highlight the potential interest of SVPs to control this pest, shedding light on how to ultimately develop improved target-specific formulations.