BACKGROUND/OBJECTIVES: To assess the efficacy of dynamic intraoperative spectral-domain optical coherence tomography (iSD-OCT) imaging for inverted internal limiting membrane (ILM) flap technique (IILMFT) in large macular hole (MH) surgery. SUBJECTS/METHODS: Prospective, non-randomized, observational study was conducted on 8 eyes of 7 patients with large, chronic and recurrent MHs, which were treated by pars plana vitrectomy (PPV) with IILMFT. All patients underwent standard pre- and postoperative examination. The iSD-OCT imaging was performed using microscope integrated systems before, during, and after ILM peeling. The iSD-OCT data were post-processed using graphic software and reviewed for tissue behavior and instruments position. RESULTS: The real-time iSD-OCT-assisted IILMFT allowed for real-time imaging of the entire surgery with visualization of the MH, vitreoretinal instruments, and all steps of inverted ILM flap formation. In spite of shadowing created by the steel instruments, it was possible to follow and control the distance between the instrument tips and retinal layers. Dynamic imaging of the surgical maneuvers including ILM peeling and mechanical apposition of MH edges revealed the iatrogenic impact on the retina (depression and appearance of hyporeflective zones). iSD-OCT imaging could confirm the proper position of the inverted ILM flap at the very end of the surgery after fluid-air exchange. CONCLUSIONS: iSD-OCT imaging is an effective tool for learning and performing a well-controlled and safe inverted ILM flap technique in patients with large MH. Clinical significance of the structural iSD-OCT findings has to be further studied.