The study develops a microfluidic metastasis-on-chip assay to model early stages of cancer metastasis, specifically focusing on triple-negative breast cancer (TNBC) using the MDA-MB-231 cell line. The assay integrates stromal fibroblasts and a perfused microvessel to recapitulate the tumor microenvironment (TME). High-content imaging and automated quantification methods were optimized to assess tumor cell invasion and intravasation. The presence of fibroblasts significantly enhanced tumor cell invasion and intravasation, while the non-invasive MCF7 cell line remained non-invasive. A targeted anti-cancer drug library was screened, identifying 30 compounds that reduced tumor intravasation by 60% compared to controls. Multi-parametric phenotypic analysis revealed that the drug library clustered into eight distinct groups, with MEK inhibitors showing strong effects on cell invasion and intravasation. Imatinib, a multi-kinase inhibitor, was found to enhance endothelial barrier stability and reduce tumor cell intravasation. The findings demonstrate the potential of the metastasis-on-chip assay as a powerful tool for studying cancer metastasis biology and drug discovery, offering prospects for personalized anti-metastatic therapies for TNBC patients.The study develops a microfluidic metastasis-on-chip assay to model early stages of cancer metastasis, specifically focusing on triple-negative breast cancer (TNBC) using the MDA-MB-231 cell line. The assay integrates stromal fibroblasts and a perfused microvessel to recapitulate the tumor microenvironment (TME). High-content imaging and automated quantification methods were optimized to assess tumor cell invasion and intravasation. The presence of fibroblasts significantly enhanced tumor cell invasion and intravasation, while the non-invasive MCF7 cell line remained non-invasive. A targeted anti-cancer drug library was screened, identifying 30 compounds that reduced tumor intravasation by 60% compared to controls. Multi-parametric phenotypic analysis revealed that the drug library clustered into eight distinct groups, with MEK inhibitors showing strong effects on cell invasion and intravasation. Imatinib, a multi-kinase inhibitor, was found to enhance endothelial barrier stability and reduce tumor cell intravasation. The findings demonstrate the potential of the metastasis-on-chip assay as a powerful tool for studying cancer metastasis biology and drug discovery, offering prospects for personalized anti-metastatic therapies for TNBC patients.