Significance: Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothrombosis model, the photosensitizer rose bengal (RB) is systemically introduced into the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of RB is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific, especially in the depth dimension. We developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. Aim: We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of RB to confine photothrombosis within a vessel of a target depth. Approach: FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), RB is injected into the blood stream. The scanning wavelength is changed to 720 nm, and photothrombosis is induced by scanning the target vessel. Results: Two-photon depth-targeted single-vessel photothrombosis was achieved with a success rate of 84.9 % ± 1.7 % and an irradiation duration of < 80    s . Attempts without RB (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. Conclusions: We described a protocol that achieves depth-targeted single-vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.