Abstract Background/aim: A growing body of research is contributing to the development of three-dimensional (3D) tissue models to close the gap between two-dimensional (2D) cell culture and animal models. Here, we report fundamental studies to confirm the modification of vascular endothelial (VE)-cadherin by a tumor microenvironment using 2D and 3D in vitro models of triple-negative breast cancer cells co-cultured with endothelial cells. Materials and methods: Breast cancer cells were cultivated as a monolayer (2D) on plates for 5 days or as microtumor spheroids (3D) with endothelial cells for up to 6 days. Phosphotyrosine-containing protein panels were analyzed in both cell types and upon co-culture. Microtumor spheroid size was evaluated via phase contrast microscopy. The content of VE-cadherin and phospho-VE-cadherin was determined. The effect of microtumor spheroid on the capillary network formed by endothelial cells was quantified by ImageJ Angiogenesis Analyzer. Sunitinib was used to determine drug efficacy in this model. Results: The activity of signaling pathways in endothelial cells, including phosphorylation of Y685-VE-cadherin, was increased by the presence of breast cancer cells. In the 3D co-culture system, we established a ratio of the two cell types which allowed viability for 6 days. As a proof-of-concept of the 3D co-culture system for the process of drug discovery and development, we used the system to quantify the efficacy of sunitinib on the phosphorylation of VE-cadherin. Conclusion: In summary, we established 2D and 3D breast cancer-endothelial cell test systems compatible for detection of minimally tyrosine-phosphorylated proteins including VE-cadherin. The systems are capable of quantifying the effect of drugs on a tissue model of angiogenesis. This is a step towards developing tools for drug-efficacy testing that do not rely on live animals. Keywords: Three-dimensional model; breast cancer cells; endothelial cells; in vivo mimicking.