Tuta absoluta Meyrick is an invasive insect pest native to South America. Because of its exceptional rapid spread and resistance to conventional insecticides, an effective control is needed. Spinosad, a natural pesticide, seems to be an alternative but few studies were done to understand the induction of the resistance process. Moreover, the delayed effects of spinosad are still unknown and must be highlighted. In this study, spinosad was tested by topical application on the last instar larvae of T. absoluta and its biological impact evaluated in two successive generations (G0 and G1) using traditionnal biomarkers of toxicity and reprotoxicity. Initially, toxicity tests were used to determine the inhibition doses (ID) of pupal moult (ID₅₀ 243.50 ng). Biochemical assays reveal a detoxification process, an oxidative stress and an inhibition of acetylcholinesterase in G0 and G1. In addition, our data show a decrease in vitellogenins and vitellins content in the two generations, which may affect negatively the fertility and fecundity of the adults. For all considered parameters, we prove that spinosad acts with delayed effects on G1.