The inflammatory response in endothelial cells (ECs) leads to an increase in vascular permeability through the formation of gaps. However, the dynamic nature of vascular permeability and external factors involved is still elusive. In this work, we use a biomimetic blood vessel (BBV) microfluidic model to measure in real-time the change in permeability of the EC layer under culture in physiologically relevant flow conditions. This platform studies the dynamics and characterizes vascular permeability when the EC layer is triggered with an inflammatory agent using tracer molecules of three different sizes, and the results are compared to a transwell insert study. We also apply an analytical model to compare the permeability data from the different tracer molecules to understand the physiological and bio-transport significance of endothelial permeability based on the molecule of interest. A computational model of the BBV model is also built to understand the factors influencing transport of molecules of different sizes under flow. The endothelial monolayer cultured under flow in the BBV model was treated with thrombin, a serine protease that induces a rapid and reversible increase in endothelium permeability. On analysis of permeability data, it is found that the transport characteristics for fluorescein isothiocyanate (FITC) dye and FITC Dextran 4k Da molecules are similar in both BBV and transwell models, but FITC Dextran 70k Da molecules show increased permeability in the BBV model as convection flow (Peclet number > 1) influences the molecule transport in the BBV model. We also calculated from permeability data the relative increase in intercellular gap area during thrombin treatment for ECs in the BBV and transwell insert models to be between 12% and 15%. This relative increase was found to be within range of what we quantified from F-actin stained EC layer images. The work highlights the importance of incorporating flow in in vitro vascular models, especially in studies involving transport of large size objects such as antibodies, proteins, nano/micro particles, and cells.