Complex biological or environmental signaling events can lead to cell migration, the movement of cells from one location to another. The migration of cells commonly occurs during both normal biological processes such as the development of an organism or marking pathological events such as cancer metastasis. Automated processes in higher density formats improve throughput greatly increasing the usefulness of performing migration assays at a larger scale. This study evaluated the use of a high density cell exclusion assay in 384- well format for investigation of cell migration using a cell line stably expressing green fluorescent protein (GFP).