This dataset contains raw and processed data associated with the research article by Infante O et al., "Insights into the structural changes that trigger receptor binding upon proteolytic activation of Bacillus thuringiensis Vip3Aa insecticidal protein." The dataset includes results from ELISA assays (ELISA assays.zip), and image-based segmentation for cell mortality analyses (Imaging assays.zip), corresponding to Figures 1D, 2, 3C, 4C, and S1 from the paper. The dataset is organized into two zip files: Absorbance Data (xlsx format): This file contains the results of ELISA binding saturation assays related to Figures 1D, 3C, 4C, and S1. Data Figure 1D: ELISA binding saturation assay data for Vip3Aa protoxin and activated toxin binding to S. frugiperda BBMV. Data Figure 3C: ELISA binding saturation data for Vip3Aa fragments binding to S. frugiperda BBMV. Data Figure 4C: ELISA binding saturation data for Vip3Aa mutants binding to S. frugiperda BBMV. Data Figure S1: ELISA data showing the recognition of Vip3Aa protoxin and activated toxin by anti-Vip3Aa antibody. Images and Data for Figure 2 (images and text files): This file contains the source images, processed images, data files, and plots used to create Figure 2. These data are generated using the Jupyter notebooks available at https://github.com/adanog/Vip3Aa. Source Images: Raw images of Sf9 cells captured 72 h post-treatment with Vip3Aa or Cry1Fa protoxin or activated toxin. Segmented Images: Processed images of cells segmented using Cellpose3. Data Files: Text files containing data on cell mortality and cell size changes, based on the reduction in cell count and increases in cell size relative to the controls. Plots: Graphical representations of cell mortality and size changes used in Figure 2. Scripts (Available in GitHub as Jupyter Notebooks): The Jupyter notebooks for cell segmentation and analysis, which generate the images, data, and plots for Figure 2, are available at: https://github.com/adanog/Vip3Aa. The scripts include: cyto3_edited.ipynb: Segments and outlines cells in the source images, producing segmented images. Dose_response_analysis.ipynb: Analyzes cell mortality and generates the associated data files and plots. Plot_dose_response.ipynb: Calculates changes in cell size and generates the data files and plots for cell swelling analysis.