Subretinal injection is performed in vitreoretinal surgery with two main aims, namely, the subretinal delivery of therapeutic agents and subretinal injection of fluid to induce a controlled and localized macular detachment. The growing interest in this technique is mainly related to its suitability to deliver gene therapy in direct contact with target tissues. However, subretinal injection has been also used for the surgical management of submacular hemorrhage through the subretinal delivery of tissue plasminogen activator, and for the repair of full-thickness macular holes, in particular refractory ones. In the light of the increasing importance of this maneuver in vitreoretinal surgery as well as of the lack of a standardized surgical approach, we conducted a comprehensive overview on the current indications for subretinal injection, surgical technique with the available variations, and the potential complications.