<p>This study aimed to assess the applicability of the Orbit^® Subretinal Delivery System [Orbit^® SDS, 2nd Generation, clinical-grade device] and prototypes for subretinal injection (SRI) in dogs of different ages. Following ex vivo and in vivo testing of the clinical grade Orbit^® SDS in 7 adult canine eyes, 8 normal dogs (13 eyes; age range: 10–72 weeks) were injected with an AAV2/5-CBA-tdTomato-WPRE vector using different Orbit^® SDS prototype devices. Dogs underwent non-invasive fundus photography regularly post-injection (PI). Retinal imaging including optical coherence tomography (OCT) angiography (OCTA) and <i>en face</i> null OCT was performed in most dogs at 5- and 9-weeks PI. Following termination, eyes were examined histologically. Ex vivo and in vivo SRI could be performed in 7 adult eyes including in that of a dog with end stage retinal degeneration. SRI of AAV2/5-CBA-tdTomato-WPRE was also successful in all 13 eyes including those of juvenile animals, yet this frequently required multiple attempts and/or using custom-modified devices that were adjusted to the smaller curvature of the ocular globes. Localized tdTomato expression was found in 11/13 eyes along the cannula path in the suprachoroidal space (SCS) in the sclera and choroid. Clinical and cellular signs of inflammation were seen in 7/13 eyes. The Orbit^® SDS device allows for controlled subretinal injections via a posterior approach in adult and juvenile dogs. There was variable vector reflux from the subretinal bleb and/or leakage from the device into the SCS. A cellular immune reaction to the AAV capsid/transgene may have contributed to retinal and choroidal inflammation.</p>