Cytoskeletal proteins function as dynamic and complex components in many aspects of cell physiology and the maintenance of cell structure. However, very little is known about the coordinated system of these proteins. The knowledge of subcellular localization of proteins is crucial for understanding how proteins function within a cell. We present a framework for quantification of cytoskeletal protein localization from high-content microscopic images. Analyses of high content images of cells transfected by cytoskeleton genes involve individual cell segmentation, intensity transformation of subcellular compartments, protein segmentation based on correlation coefficients, and colocalization quantification of proteins in subcellular components. By quantifying the abundance of proteins in different compartments, we generate colocalization profiles that give insights into the functions of different cytoskeletal proteins.