The glycocalyx of endothelial cells is a dynamic, gel-like layer of glycoproteins, proteoglycans, and glycolipids that lines the luminal surface of blood vessels, playing a critical role in vascular permeability, mechanotransduction, and protection against shear stress. In this study, we investigated the in vitro adhesion of giant unilamellar vesicles (GUVs) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dimyristoyl--glycero-3-phosphocholine (DMPC). Specifically, we examined mildly positively charged DOTAP-DMPC (20:80) GUVs, based on positively charged DOTAP and neutral DMPC but exhibiting an overall mild positive charge in physiological buffer, and neutral DMPC GUVs, which show a negative charge in physiological buffer. Adhesion to human umbilical vein endothelial cells (HUVEC) was studied under three culture conditions: dynamic (intact glycocalyx), static (underdeveloped glycocalyx), and glycocalyx-shed (degraded glycocalyx). Vesicles were produced via electroformation, stained with Texas Red dye, and perfused over endothelial cells at a controlled velocity to simulate slow blood flow. Adhesion was tracked using fluorescence microscopy combined with cell segmentation techniques. Adhesion of DOTAP-DMPC vesicles was significantly enhanced-by approximately 3.5-fold-on glycocalyx-shed cells compared to cells with an intact glycocalyx. In contrast, DMPC vesicles showed no adhesion under any condition. Analysis of vesicle size distributions revealed no significant differences between adherent and nonadherent vesicles or between DOTAP-DMPC and DMPC vesicles. These findings provide insights into the role of the endothelial glycocalyx in regulating adhesion, with potential implications for tumor cell interactions with the endothelium and mechanisms underlying DOTAP-based transfection.