Focal adhesions provide a linkage between an anchorage dependant cell and extracellular matrix (ECM) and mediate cell functions (eg. migration). Our understanding of the focal adhesion regulation of cell behavior, however, has been limited due to the involvement of multiple proteins and their complex network of interactions. The present study is an effort to unfold this complexity by mapping the dynamic organization of focal adhesion proteins (eg. vinculin and paxillin) in fibroblasts using immunofluorescent imaging techniques. Specifically, we explored two main questions: 1) how do substrate properties (eg. ligand density) regulate the spatial and temporal dynamics of focal adhesions? and 2) what are the potential mechanisms of the focal adhesion regulation of cell spreading, shape and motility? To answer these questions, we developed metrics to quantify focal adhesions and cell spreading. We demonstrate that these metrics delineate the effects of cell-ECM interactions on focal adhesion formation.