Cancer is a frequent disease in western countries and there is no effective treatment for metastasis, the main cause of death in cancer patients. The situation can be improved by a better understanding of the cancer invasion process. In order to reveal new aspects of this dynamic process, we developed a novel direct viewing cancer cell invasion assay with shear flow in vitro. This assay comprised of a custom-made flow chamber, specially developed cell labelling, high-resolution wide-field microscopy and image-processing-based quantitation. We applied this assay to metastatic rat sarcoma cells which invaded monolayers of rat endothelial cells. Our findings showed that after adhesion, the sarcoma cells initially invaded significantly faster under flow conditions compared to situations without shear stress. Later, however, the rate of invasion under flow decreased and the sarcoma cells without shear stress achieved significantly higher levels of invasion. Our observations thus revealed the non-linear modulation of a cancer cell invasion process by shear flow, demonstrating that cancer cells can respond to flow by enhancement of invasiveness similarly to white blood cells.