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Lamellar techniques for selective replacement of diseased corneal structures have recently been improved. Descemet membrane endothelial keratoplasty (DMEK) allows the sole replacement of the endothelium-Descemet membrane layer (EDM). However, widespread use of DMEK is currently limited because of problems with donor preparation namely the tearing of the Descemet membrane and the difficulty to unfold the EDM graft in the anterior chamber (AC).A standardized DMEK procedure that allows safe preparation of EDM, atraumatic introduction of EDM into the AC, reliable orientation of EDM during surgery, and stepwise unfolding within the AC is described in 80 patients. Visual acuity and corneal endothelial cell density were assessed.A stepwise approach using a novel bimanual underwater technique to harvest EDM from donor corneal buttons allows reproducible generation of grafts without tearing the Descemet membrane. Injection of the EDM roll into the AC is achieved by use of a standard injector cartridge, whereas the depth of AC is maintained by an irrigation handpiece. Marks at the margin of EDM allow orientation. Finally, unfolding EDM in the AC is achieved by sequential use of water jets and air bubbles. In the early phase of the learning curve, 4 patients were regrafted because of graft failure. Endothelial cell density decreased from 2600 ± 252 to 1526 ± 341 cells per square millimeter 1 month after DMEK.A novel technique for graft preparation and EDM injection results in improved safety with a high rate of successful DMEKs.