Bacillus thuringiensis is a Gram-positive spore- forming bacterium known for its insecticidal activities. Although its features of high virulence and exceptional host specificity are wiely known and have conditioned its use as a source of novel biopesticides, molecular mechanisms underlying these traits remain elusive and are usually attributed to its repertoire of proteinaceous toxins. In this work we used combined proteogenomic approach to dissect <>. We used three different strains of B. thuringiensis belonging to the var. thuringiensis, darmstadtiensis and israeliensis and one derivate of B. thuringiensis var israeliensis, which lost the ability to produce Cry-toxins. By using hybrid Oxford Nanopore and Illumina sequencing we achieved replicon-level genome assemblies of the studied strains which, upon annotation, facilitated comparison of virulence factor repertoires and putative reasons of virulence loss in derivate strain, Further application of quantitative HPLC-Orbitrap-MS and proteome level-com