The ability to view and record the movements of subcellular structures is a powerful tool that has accelerated the discovery and understanding of signaling mechanisms that control microvascular functions such as the control of endothelial permeability. Advances in molecular biology over the past few decades have facilitated the generation of fusion proteins in which fluorescent reporters based upon the structure of green fluorescent protein can be linked to proteins found in human endothelial cells, such as VE-cadherin or β-actin. These fusion proteins have been found to incorporate into structures alongside their native protein counterparts, allowing the dynamic visualization of how these subcellular structures are modified when cells are challenged with stimuli such as inflammatory mediators. The result of such studies has been a much more advanced view of the complex mechanisms by which endothelial cells maintain barrier properties than previously obtained by only viewing fixed cells labeled by immunofluorescence. Here, we describe our protocols that we have used to view the dynamics of actin filaments using time-lapse microscopy to record endothelial cells expressing GFP-actin and the analysis tools available to quantify dynamics of subcellular structures.