Of the known risk factors for glaucoma, elevated intraocular pressure (IOP), is the primary one. The conventional aqueous humor outflow pathway contains the key source of IOP regulation, which is predominantly the trabecular meshwork (TM). Studies of outflow have demonstrated that the outflow pathway is not uniform around the circumference of the eye but highly segmental with regions of relative high flow (HF) and intermediate or medium flow (IF) and regions of low or no flow (LF). Herein we present protocols that we use to study outflow segmentation through the conventional outflow pathway, mostly focusing on human eyes. These methods are quite similar for nonhuman primates and other species. These studies are mostly conducted using ex vivo intact globes or perfused anterior segment organ culture. One potential therapy for IOP reduction in those with elevated IOP to reduce progression of glaucomatous optic nerve damage would be to increase HF or IF and reduce LF proportions.