Brush border membrane vesicles (BBMV) from insect midguts are an in vitro model for studying Bacillus thuringiensis (Bt) toxins. Despite their widespread use, the protein components of these vesicles are mostly unknown. Two-dimensional electrophoresis (2DE) was used to analyze vesicles from Manduca sexta and establish two subproteomes: GPI-anchored proteins and Cry1Ac binding proteins. Through mass spectrometry and western blot analyses, two new Bt binding proteins were identified from 2DE blots: alkaline phosphatase and actin. BBMV proteins from diet adapted Bt-susceptible and -resistant Plutella xylostella strains were tagged with fluorescent dyes and simultaneously compared by two dimensional difference gel electrophoresis (2D-DIGE). Although BBMV derived from whole animals are a suitable substitute for BBMV prepared from dissected guts, both preparation types were analyzed by 2D-DIGE to determine if protein differences between strains were observed. More proteins were resolved in whole animal preparations, but more differences in protein abundance were detected in dissected gut BBMV preparations between susceptible and resistant P. xylostella. Mass spectrometry analysis by MALDI-ToF/ToF of several altered spots resulted in the identification of two proteins. In resistant animals, actin was identified as a less abundant protein and glucosinolate sulphatase (GSS) as a more prevalent protein. Western blot comparisons of diet-adapted and cabbage-reared susceptible and resistant animals revealed that GSS was more abundant in diet adapted strains and in resistant animals. Enzyme activity assays revealed that alkaline phosphatase levels were also higher in these populations. The amount of aminopeptidase protein was unchanged between the four P. xylostella BBMV preparations. The identification of new Bt binding proteins and proteins implicated in Bt resistance broadens the understanding of Bt action in insect midgut epithelial cells, especially post-binding interactions that lead to the insects death. This knowledge will help prolong the use of Bt as an effective biological control. Our findings realized the potential of 2DE-based proteomics as an approach for identifying proteins in insect tissues, specifically those involved in Bt action. Additionally, my studies are the first to describe the use of additional techniques, western blotting and bioinformatics analyses, to facilitate proteins identification by mass spectrometry and overcome the limitations of this technique.