Chrysomelina larvae possess a sophisticated strategy in terms of chemical defense adapted to their natural habitat. In case of a predatory attack, the deterrent secretions are presented from dorsal glands. Three different ways to produce the repellent glucosides are proposed. Irrespective of the deterrent production, all larvae show common principles: the glucosidically bound precursors are transferred via the hemolymph into the glandular reservoir. Until recently, almost no genomic and transcriptomic data were available for leaf beetles. In order to comprehensively study the defense metabolism with respect to transport proteins in leaf beetle species, the transcriptomes of Phaedon cochleariae as well as Chrysomela populi were sequenced by applying next-generation sequencing technologies. The transcript sequences of these leaf beetles were assembled de novo to provide essential transcript catalogues. These sequences were then annotated and putative functions assigned. Thereafter, in addition to phylogenetic studies, RNA interference (RNAi) methodology was applied to characterize particular enzymes and proteins. To circumvent the knock-down of non-target transcripts, an off-target prediction method was implemented. Combining RNA-seq with RNAi, and subsequently studying differential expression, promoted the observation of enzymes involved in the sequestration process. Two enzymes namely a salicyl alcohol oxidase in C. populi and a juvenile hormone-binding protein of P. cochleariae were observed, RNAi targeting the most abundant sugar transporters in P. cochleariae revealed a counter-regulation which ensures the defense ability, and RNAi targeting the most abundant glandular ABC transporter in C. populi validated its significant function. In conclusion, sequencing of transcriptomes and RNA-seq after triggering RNAi followed by the analysis of differential gene expression holds great opportunities for the study of genes of interest in non-model organisms.