Endothelial cell junctions are constantly remodelled during inflammation, angiogenesis and wound healing but the molecular background remains poorly understood. Recent advances in the developments of fluorescence tagged protein-technologies and -expression together with fast speed microscopic techniques such as spinning disc microscopy allows visualizing the dynamic remodelling directly with high spatial and temporal resolution over long time periods. We expressed VE-cadherin-mCherry fusion protein in human umbilical vein endothelial cells by lentiviral gene transfer. The fusion protein was functionally active and localized at cell junctions, and time-lapse movies were acquired by spinning disc microscopy. To analyse the reorganisation of VE-cadherin-mCherry quantitatively we developed a new software tool. In a first step the algorithm tracks the cell-borders of all cells within the image. Since it distinguishes between free cell borders and cell junctions, it is possible to follow junction dynamics in both sparse cell cultures and highly confluent endothelial cell layers. In a second step we used the segmentation to determine changes in protein concentration and dynamics in defined regions along the junctions. The analysis reveals a thrombin-dependent decrease in VE-cadherin dynamics at the cell junctions, a result that could be confirmed by fluorescence recovery after photobleaching (FRAP). Thus, the developed image analyses software allows segmentation and quantitative analyses of protein dynamics at endothelial cell junctions.