Recent developments in quantitative image analysis allow us to interrogate confocal microscopic images to answer biological questions. Clumped and layered cell nuclei and cytoplasm in confocal microscopic images challenges the ability to identify subcellular compartments. To date, there is no perfect image analysis method to identify cellular cytoskeletal changes from confocal microscopic images. Here we present a multi-disciplinary study where an image analysis model was developed to allow quantitative measurements of changes in the cytoskeleton of cells with different maspin exposure. Maspin, a non-inhibitory serpin influences cell migration, adhesion, invasion, proliferation and apoptosis in ways that are consistent with its identification as a tumor metastasis suppressor. Using different cell types we tested the hypothesis that the reduction of cell migration by maspin would be reflected in the architecture of the actin cytoskeleton. A hybrid marker controlled watershed segmentation technique was used to segment the nuclei, cytoplasm and ruffling regions prior to measuring cytoskeletal changes. This was informed by immunohistochemical staining of cells transfected stably or transiently with maspin proteins, or with added bioactive peptides or protein. Image analysis results showed that the effects of maspin were mirrored by effects on cell architecture, in a way that could be described quantitatively.