The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the in vivo process. The most commonly used in vitro invasion assay is a modified Boyden chamber assay using a basement membrane matrix preparation, Matrigel, as the matrix barrier and NIH-3T3 conditioned media as the chemoattractant. The results obtained using this assay show a strong correlation between the ability of tumor cells to invade in vitro and their invasive behavior in vivo, which validates this assay as a measure of invasive potential. The methods presented in this chapter outline how the Matrigel in vitro invasion assay is performed.