Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and analysis of their colocalization. Here we present the applications Squassh3C and SquasshAnalyst. Squassh3C extends the functionality of Squassh to three fluorescence channels and live-cell movie analysis. SquasshAnalyst is an interactive web interface for the analysis of Squassh3C object data. It provides segmentation image overview and data exploration, figure generation, object and image filtering, and a statistical significance test in an easy-to-use interface. The overall procedure combines the Squassh3C plug-in for the free biological image processing program ImageJ and a web application working in conjunction with the free statistical environment R, and it is compatible with Linux, MacOS X, or Microsoft Windows. Squassh3C and SquasshAnalyst are available for download at www.psi.ch/lbr/SquasshAnalystEN/SquasshAnalyst.zip .