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Cell migration plays a pivotal role in many biological processes and is modulated by cytokines and growth factors. In vivo, cells are embedded in an extracellular matrix (ECM). ECM proteins are linked to the cellular cytoskeleton by integrin adhesion receptors, which transmit extracellular signals into the cell, thereby affecting cell adhesion and migration as well as gene expression. We describe a cell migration assay that uses a barrier device to separate the cells. The assay enables quantification of the migration of adherent cells on defined matrix proteins and the ability to evaluate migration-associated characteristics of individual cells. Thus, the barrier cell migration assay is a useful tool for exploring matrix-dependent migration of adherent cells.