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To describe a novel surgical technique to produce thin endothelial grafts for Descemet stripping automated endothelial keratoplasty (DSAEK).Thirteen human cadaveric corneas in organ culture were randomized into conventional (n = 7) and microthin (n = 6) DSAEK groups. Grafts in the conventional DSAEK group were prepared using the conventional DSAEK technique of a single microkeratome pass with a 350-μm cutting head. Corneas in the microthin group were preconditioned to achieve a target central thickness of 530 μm before graft dissection with a 350-μm microkeratome head. Preconditioning involved stromal dehydration under pachymetric control using sterile airflow for 15-second increments. Donor and graft thicknesses were assessed with optical coherence tomography, and endothelial viability with trypan blue and alizarin red staining.Mean endothelial graft thickness obtained using the microthin DSAEK technique was 106 μm (SD, 32 μm) compared with 177 μm (SD, 33 μm) obtained using conventional DSAEK technique (P = 0.0024). Donor preconditioning yielded a predicted reduction of 100 μm in graft stromal thickness at a rate of 1.5 μm/s and mean duration of 72 seconds. The average anterior lamella thickness (cut depth) obtained in microthin and conventional DSAEK groups were 424 and 431 μm, respectively (P = 0.84). There was no difference in endothelial viability between the 2 groups. There were no corneal perforations during graft preparation in this study.Donor preconditioning by pachymetry-controlled stromal dehydration achieved significantly thinner endothelial grafts compared with the conventional DSAEK technique without compromise to endothelial viability or graft wastage.