Assays that conveniently quantify invasion of carcinoma cells in vitro have generally measured the passage of dissociated cells into a matrix. Although these assays have been helpful in identifying relative differences between different carcinoma cell lines or types, the requirement for dissociation overlooks the possible modulation of invasion by cell-cell interactions among carcinoma cells. Described here is a novel assay that quantifies invasion of a matrix placed above intact, multilayered raft cultures of lung carcinoma cell lines A549 and H520. The assay was performed by placing a porous membrane coated with matrix at the air interface of the raft cultures for varying lengths of time, after which the cells invading the matrix were enumerated. The numbers of cells invading increased in a relatively linear fashion from 24 to 72 h, and the absolute numbers within each cell line were reproducible with multiple sets of raft cultures prepared at different times. It was also found that this assay could quantify differences in invasion caused by changes in matrix composition. It is concluded that this assay can reproducibly quantify carcinoma cell invasion from three dimensional raft cultures.