We report the design and synthesis of an activatable molecular imaging probe to detect hypoxia in mouse models of retinal vascular diseases. Hypoxia of the retina has been associated with the initiation and progression of blinding retinal vascular diseases including age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. In vivo retinal imaging of hypoxia may be useful for early detection and timely treatment of retinal diseases. To achieve this goal, we synthesized HYPOX-3, a near-infrared (NIR) imaging agent coupled to a dark quencher, Black Hole Quencher 3 (BHQ3), which has been previously reported to contain a hypoxia-sensitive cleavable azo-bond. HYPOX-3 was cleaved in hypoxic retinal cell culture and animal models, enabling detection of hypoxia with high signal-to-noise ratios without acute toxicity. HYPOX-3 fluorescences in hypoxic cells and tissues and was undetectable under normoxia. These imaging agents are promising candidates for imaging retinal hypoxia in preclinical disease models and patients.