Clinical Relevance: The endothelial monolayer is a dynamic, single cell layer which forms a barrier to prevent blood fluids from leaking into the surrounding tissues. During inflammation, endothelial barrier disruption causes vascular leakage which contributes to disease severity in disorders such as sepsis, cancer, diabetes, and atherosclerosis. Despite this tremendous medical threat to the critically ill patients, specific therapy counteracting vascular leakage are lacking up to today. Rationale: Hyperpermeability of the endothelial barrier is a significant pathogenic phenomenon and is associated with many life-threatening diseases. Previous studies have shown that contractile forces, generated by the endothelium itself, play an important role in the process of barrier dysfunction. With the use of traction force microscopy it is possible to study the developed acto-myosin dependant interaction via the focal adhesions to the cells substrate. However, a measurement of the situation at endothelial cell-cell junctions was not experimentally accessible. Here, we aim for the first time to elucidate the intercellular forces in an endothelial monolayer sheet and observe the developed stresses during thrombin receptor-mediated hyperpermeability. Methods & Results: Endothial cell monolayer contractions were mapped by the novel described monolayer stress microscopy (1) in combination with previous published traction force microscopy of 4 kPa polyacrylamide gel substrates. (2) Displacements of the caroxylate-modified fluorescent microspheres (Invitrogen, Eugene, OR) incorporated in the gels, were converted into stress components and monolayer traction forces. Conclusion: Novel intracellular stress mapping in endothelial monolayer was successful and displaced highly diverse stress maps which provides new information about vascular hyperpermeability responses.