Protein baits containing fipronil as a biocide have shown their effectivity as a control method for Vespa velutina nigrithorax (Lepeletier, 1836) in apiaries. This biocide is not selective for Vespa velutina, so it is important to use the minimum dose to inactivate a nest. Therefore, the aim of this work was the development of analytical methods for the determination of fipronil in protein baits for quality control purposes and in larvae of Vespa velutina to determine the biocide content after protein bait ingestion and to acquire knowledge on fipronil metabolism in larvae. For this purpose, a Quechers-based HPLC-PDA method was developed and validated for the determination of fipronil in both matrixes. Furthermore, a GC-MS method was developed for the analysis of fipronil and its metabolites in dead Vespa velutina larvae fed with a mash containing 0.01% fipronil. Quechers-based HPLC-DAD allowed for the determination of the fipronil content in baits. Fipronil and the metabolites fipronil sulfone and fipronil sulfide were identified by GC-MS in extracts of larvae fed with a protein mash containing 0.01% fipronil. The transformation of fipronil into fipronil sulfone inside the larvae and the high toxicity of this metabolite open the possibility to produce protein baits with lower biocide concentrations.