To evaluate clinical outcomes of Descemet Membrane Endothelial Keratoplasty (DMEK) using a novel hydro-separation of donor Descemet membrane (DM) endothelial graft with 6 months follow up. In this retrospective single-center; single surgeon cohort study, 16 consecutive eyes underwent DMEK or combined DMEK and cataract surgery with DMEK donor graft hydro-separation. The hydroseparation method used Ringer’s solution injected by a syringe with a flat end cannula between DM and corneal stroma to separate DMEK graft from donors’ cornea. Endothelial cell count, corrected and uncorrected distance visual acuities (CDVA, UDVA), corneal astigmatism and keratometric values were evaluated before and 6 months after the surgery. DMEK grafts were successfully hydro separated, without a membrane tear in all cases and successful adhesion to recipient corneas was achieved in all cases. Overall CDVA improved significantly (p < 0.0001) from preop.0.69 logMAR (0.45–0.92, 95%CI) to 0.1 logMAR (0.04–0.15) 6 months postop. However UDVA was slightly worse in cataract combined cases 0.38 logMAR (0.18–0.43) vs 0.3 logMAR (0.14–0.61) in DMEK cases at 6 months. Corneal astigmatism K2-K1 improved significantly (p=0.0137) from preop. mean 2.89 D (0.19–0.43) to 0.98 D (0.48–1.48) 6 months postop. Hydro-separation is fast and effective donor graft preparation method for DMEK. The DMEK using donor graft hydro-separation results in improved vision and induces a low amount of astigmatism at 6 months follow up. Further clinical data are needed to confirm the success rate and to explore optimization of the selection of IOLs in combined cataract cases