Selective Large-Area Retinal Pigment Epithelial Removal by Microsecond Laser in Preparation for Cell Therapy Cell therapy is a promising treatment for retinal pigment epithelium (RPE)-associated eye diseases such as age-related macular degeneration. Herein, selective microsecond laser irradiation targeting RPE cells was used for minimally invasive, large area RPE removal in preparation for delivery of retinal cell therapeutics. Ten rabbit eyes were exposed to laser pulses 8, 12, 16, and 20 μs in duration (wavelength, 532 nm; top-hat beam profile, 223 × 223 μm2). Post-irradiation retinal changes were assessed with fluorescein angiography (FA), indocyanine green angiography (ICGA), and optical coherence tomography (OCT). RPE viability was evaluated with an angiographic probit model. Following vitrectomy, a subretinal injection of balanced salt solution was performed over a lasered (maximum 13.6 mm2) and untreated control area. Bleb retinal detachment (bRD) morphology was then evaluated by intraoperative OCT. Within 1 hour after irradiation, laser lesions showed FA and ICGA leakage. OCT revealed that large-area laser damage was limited to the RPE. The angiographic median effective dose irradiation thresholds (ED50) were 45 μJ (90 mJ/cm2) at 8 μs, 52 μJ (104 mJ/cm2) at 12 μs, 59 μJ (118 mJ/cm2) at 16 μs, and71 μJ (142mJ/cm2) at 20 μs. Subretinal injection over the lasered area resulted in a controlled, shallow bRD rise, whereas control blebs were convex in shape, with less predictable spread. Large-area, laser-based removal of host RPE without visible photoreceptor damage is possible and facilitates surgical retinal detachment. Selective microsecond laser-based, large-area RPE removal prior to retinal cell therapy may reduce iatrogenic trauma.