Primary open-angle glaucoma (POAG) is a leading cause of blindness in the world. A primary risk factor for POAG is elevated intraocular pressure (IOP), caused by increased aqueous humor outflow resistance. Currently, lowering the IOP is the only effective way of treating glaucoma; however, the cause of increased outflow resistance remains unclear. This thesis will present a series of studies which investigated structures of the trabecular outflow pathway, including Schlemm’s canal endothelium, juxtacanalicular tissue, and trabecular beams, and their roles in regulating aqueous outflow resistance. The studies were conducted in both human and animal models using ex vivo ocular perfusion as well as in vitro microfluidic systems. In the first study, we investigated the effects of Y27632, a derivative of Rho-kinase inhibitor that is being developed as next generation glaucoma drug with unclear IOP lowering mechanism, on aqueous humor outflow dynamics and associated morphological changes in normal human eyes and laser-induced ocular hypertensive monkey eyes. In the second study, we developed and validated a novel three-dimensional microfluidic system using lymphatic microvascular endothelial cells. The microfluidic system can be used to study Schlemm’s canal endothelial cell dynamics and aqueous humor transport mechanism in the future. In the last study, we characterized the morphological structure, distribution, and thickness of the endothelial glycocalyx in the aqueous humor outflow pathway of human and bovine eyes. Together these studies will help define new directions for therapy that will help control IOP and preserve vision throughout a normal life span.