PRECIS: Hemoglobin Video Imaging (HVI) provides a noninvasive method to quantify aqueous outflow (AO) perioperatively. Trabecular bypass surgery (TBS) is able to improve, and in some cases re-establish, conventional AO. PURPOSE: The purpose of this study was to use HVI to illustrate and quantify effects of TBS on AO through the episcleral venous system. DESIGN: This is a prospective observational cohort study. PARTICIPANTS: Patients were recruited from Sydney Eye Hospital, Australia. The study included 29 eyes from 25 patients, 15 with glaucoma and 14 normal controls. TBS (iStent Inject) was performed on 14 glaucomatous eyes (9 combined phacoemulsification/TBS and 5 standalone TBS). Cataract surgery alone was performed on the remaining eye from the glaucoma group and 2 eyes from the control group. METHODS: We used HVI, a novel clinic-based tool, to visualize and quantify AO perioperatively during routine follow-up to 6 months. Angiographic blood flow patterns were observed within prominent aqueous veins on the nasal and temporal ocular surface. Aqueous column cross-section area (AqCA) was compared before and after surgery. MAIN OUTCOME MEASURES: AqCA, number of aqueous veins, intraocular pressure (IOP) before and after surgery, and number of IOP-lowering medications. RESULTS: Patients with glaucoma had reduced AqCA compared with normal controls (P=0.00001). TBS increased AqCA in 13 eyes at 1 month (n=14; P<0.002), suggesting improved AO. This effect was maintained at 6 months in 7 eyes (n=9, P≤0.05). All patients with unrecordable AO before surgery (n=3; 2 standalone TBS, 1 combined cataract/TBS) established measurable flow after TBS. IOP and/or medication burden became reduced in every patient undergoing TBS. Cataract surgery alone (n=3) increased AqCA in nasal and temporal vessels at 4 weeks after surgery. CONCLUSIONS: HVI provides a safe method for detecting and monitoring AO perioperatively in an outpatient setting. Improvement of AO into the episcleral venous system is expected after TBS and can be visualized with HVI. TBS is able to improve, and in some cases re-establish, conventional AO. Cataract surgery may augment this. Some aqueous veins were first seen after TBS and these patients had unstable postoperative IOP control, which possibly suggests reorganization of aqueous homeostatic mechanisms. HVI may confirm adequacy of surgery during short-term follow-up, but further work is required to assess the potential of HVI to predict surgical outcomes and assist with personalized treatment decisions.