Macrophages orchestrate multiple functions in the tumor microenvironment leading to metastatic progression including promotion of tumor cell invasion, intravasation, and extravasation at metastatic sites. In vitro assays have been developed to explore these processes in high resolution. Here we describe an in vitro protocol used to examine macrophage enhanced tumor cell extravasation and intravasation in 3D using a stratified co-culture transendothelial migration (TEM) transwell system. These assays may be generally applied in order to study different aspect of metastatic dissemination, and we show here usefulness with breast and pancreatic tumor cell lines.