Abstract Motivation: In order to obtain statistically relevant results, the study of membrane effects at the single-vesicle level requires the analysis of several hundreds of giant unilamellar vesicles (GUVs), which becomes a very time-consuming task if carried out manually. Complete and user-friendly software for fast and bias-free automated analysis has not been reported yet. Results: We developed a framework for the automated detection, tracking and analysis of individual GUVs on digital microscopy images. Our tool is suited to quantify protein binding to membranes as well as several aspects of membrane permeabilization on single vesicles. We demonstrate the applicability of the approach by comparing alternative activation methods for Bax, a pore-forming protein involved in mitochondrial permeabilization during apoptosis. Availability and Implementation: The complete software is implemented in MATLAB (The MathWorks, Inc., USA) and available as a standalone as well as the full source code at http://www.ifib.uni-tuebingen.de/research/garcia-saez/guv-software.html. Contact: ana.garcia@uni-tuebingen.de Supplementary information: Supplementary data are available at Bioinformatics online.