Although the vasculature was once thought to be a passive conduit for blood, it is now known that the endothelium is responsible for healthy vascular homeostasis and the progression of many cardiovascular-related diseases. Because the endothelium lines blood vessels, it is subjected to the mechanical forces due to of blood flow. It is now well established that endothelial cells transduce these mechanical signals into chemical signals that are evident in the mechanoregulation of a number of signal transduction pathways and endothelial cell phenotype. Despite the significant volume of work in the field of endothelial cell mechanotransduction, the exact mechanism by which mechanical forces are sensed and transduced into chemical signals is not yet well established. In this chapter, we focus on the specific role of fluid shear stress, the frictional drag force caused by blood flow, and cyclic stretch caused by the pumping action of the heart, in regulating vascular homeostasis and vascular signaling. The regulation of flow-mediated signaling in the endothelium is typically studied with well-characterized in vitro flow and stretch devices. Here, we examine various platforms used to analyze flow-mediated and stretch-mediated signals and describe the method for the implementation of these techniques.