Endothelial cells that form the endothelium lining of all blood vessels are under constant shear stress from the blood flow. They have the capability of sensing differences in fluid shear stress (mechanosensing) and changing this mechanical message into a biochemical signal cascade (mechanotransduction), leading to different biological responses that could lead to cardiovascular diseases such as atherosclerosis. To understand how shear stress from blood flow is transmitted throughout endothelial cells, the magnitude and distribution of forces within each cell need to be studied. We propose a novel methodology by which we can explore cellular functions and study cytoskeleton dynamics of living cells at the subcellular level with minimal invasion. The optical tweezers technique can be used to measure the mechanical properties of the cytoskeleton in the vicinity of organelles and cellular structures. Using endocytosed silica beads as probes, we were able to determine the frequency dependent mechanical properties of the interior of cultured bovine endothelial cells. We treated the cytoskeleton with drugs that alter its composition to obtain the contribution of each cytoskeletal structure in the viscoelastic property of endothelial cells. Confocal images of drug treated cells are used as visual support for the optical tweezers results. In this talk, we will also present ongoing work on the application of the optical tweezers technique to examine the role of caveolae, which are invaginations of the plasma membrane, in mechanotransduction. Caveolae were labeled with three markers, caveolin-1, dynamin-2, and intersectin-1, in order to identify and target them as probes, and detect physical deformations in their vicinity, which are required for mechanotransduction.