MASQOT-GUI provides an open-source, platform-independent software pipeline for two-channel microarray spot quality control. This includes gridding, segmentation, quantification, quality assessment and data visualization. It hosts a set of independent applications, with interactions between the tools as well as import and export support for external software. The implementation of automated multivariate quality control assessment, which is a unique feature of MASQOT-GUI, is based on the previously documented and evaluated MASQOT methodology. Further abilities of the application are outlined and illustrated.