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The regulation of cell motility is central to living systems. Consequently, cell migration assays are some of the most frequently used in vitro assays. This article provides a comprehensive, detailed review of in vitro cell migration assays both currently in use and possible with existing technology. Emphasis is given to two-dimensional migration assays using densely organized cells such as the scratch assay. Assays are compared and categorized in an outline format according to their primary biological readout and physical parameters. The individual benefits of the various methods and quantification strategies are also discussed. This review provides an in-depth, structured overview of in vitro cell migration assays as a means of enabling the reader to make informed decisions among the growing number of options available for their specific cell migration application.