Non-invasive and high-temporal resolution methods for characterizing blood flow in mouse cranial arteries, such as the ophthalmic artery (OphA), are lacking. We present an application of pulsed Doppler ultrasound to provide real-time, non-invasive measurement of blood flow velocity in the OphA through an identified soft tissue window in the mouse head. We confirmed the identity of the artery and mapped its origin from the internal carotid artery by a combination of microcomputed tomography (microCT) vascular imaging and transient occlusion of the internal carotid artery. Application of our approach demonstrated sex differences in the OphA vasodilative response to agonists. We also evaluated real-time flow characteristics in the OphA in response to transient carotid artery ligation. The method will provide a simple and low-cost approach for screening drugs targeting ophthalmic blood flow and can be used as a more accessible surrogate of cerebral blood flow in both acute and longitudinal imaging studies.