The cellular cytoskeleton is a dynamic subcellular structure that can be a marker of key biological phenomena including cell division, organelle movement, shape changes and locomotion during the avascular tumor phase. Little attention is paid to quantify changes in the cytoskeleton while nuclei and cytoplasmic both are present in subcellular microscopic images. In this paper, we proposed a quantitative image analysis method to analyze subcellular cytoskeletal changes using a texture analysis method preceded by segmentation of nuclei, cytoplasm and ruffling regions (area except nuclei and cytoplasm). To test and validate this model we hypothesized that Mammary Serine Protease Inhibitor (maspin) acts as cytoskeleton regulator that mediates cell-extracellular matrix (ECM) adhesion in tumor. Maspin-a tumor suppressor gene shows multiple tumor suppressive properties such as increasing tumor cell apoptosis and reducing migration, proliferation, invasion, and overall tumor metastasis. The proposed method obtained separated ruffling regions from segmentation steps and then adopted gray–level histograms (GLH) and grey-level co-occurrence matrix (GLCM) texture analysis techniques. In order to verify the reliability, the proposed texture analysis method was used to compare the control and maspin expressing cells grown on different ECM components: plastic, collagen I, fibronectin and laminin. The results show that the texture parameters extracted reflect the different cytoskeletal changes. These changes indicate that maspin acts as a regulator of the cell-ECM enhancement process, while it reduces the cell migration. Overall, this paper not only presents a quantitative image analysis approach to analyze subcellular cytoskeletal architectures but also provides a comprehensive tool for the biologist, pathologist, cancer specialist, and computer scientist to understand cellular and subcellular organization of cells. In long term, this method can be extended to be used in live cell tracking in vivo, image informatics based point-of-care expert system and quantification of various complex architectures in organisms.