Abstract: Cell invasion, a hallmark of malignant cancers, plays a key role in tumor dissemination and metastasis and is responsible for cancer-related deaths. The ability of tumor cells to form a metastatic tumor primarily involves cell morphological reorganization and degradation of the surrounding extracellular matrix (ECM). Conventional in vitro invasion models, such as the filter based transwell invasion assay (2D) are widely used to assess tumor cell invasion. However, lack of comprehensive cell-ECM interactions may limit their physiological relevance. Three dimensional (3D) tumor invasion models, like the one described in this application note, potentially mimic more effectively key features of the metastatic phenotype. Understanding the mechanisms concerned in tumor cell invasion may lead to limiting tumor progression and consequently cancer patient mortality.