Endothelial cells (ECs) form the inner lining of all blood vessels in the body, and coat the outer surfaces of heart valves. Because ECs are anchored to extracellular matrix proteins and are positioned between flowing blood and underlying interstitium, ECs are constantly exposed to hemodynamic shear, and act as a semi-permeable barrier to blood-borne factors. In vitro cell culture flow (ICF) systems have been employed as laboratory tools for testing endothelial properties such as adhesion strength, shear response, and permeability. Recently, advances in microscale technology have introduced microfluidic systems as alternatives to conventional ICF devices, with a multitude of practical advantages not available at the macroscale. However, acceptance of microfluidics as a viable platform has thus far been reserved because utility of microfluidics has yet to be fully demonstrated. For biologists to embrace microfluidics, engineers must validate microscale systems and prove their practicality as tools for cell biology. Microfluidic devices were designed, fabricated, and implemented to study properties of two EC types:​ aortic ECs and valve ECs. The objective was to streamline experimentation to reveal phenotypic traits of the two types and in the process demonstrate the usefulness of microfluidics. The first task was to develop a protocol to isolate pure populations of valve ECs because reported methods were inadequate. Dispase and collagenase in combination for leaflet digestion followed by clonal expansion of cell isolates was optimal for obtaining pure valve EC populations. Using a parallel microfluidic network, we discovered that valve ECs adhered strongly and spread well only on fibronectin and not on type I collagen. In contrast, aortic ECs adhered strongly on both proteins. Both aortic and valve ECs were then exposed to shear and analyzed for cell orientation. Morphological analyses showed aortic and valve ECs both aligned parallel to flow when sheared in a macroscale flow chamber, but aortic ECs aligned perpendicular to flow when sheared in a microchannel. Finally, a microfluidic membrane device was designed and characterized as a potential tool for measuring albumin permeability through sheared endothelial monolayers. Overall, these studies revealed novel EC characteristics and phenomena, and demonstrated accessibility of microfluidics for EC studies.