Deep anterior lamellar keratoplasty (DALK) is an alternative to full-thickness corneal transplant and has advantages including the absence of allograft rejection; shortened duration of topical corticosteroid treatment and reduced associated risk of glaucoma, cataract, or infection; and enables use of grafts with poor endothelial quality. DALK begins by performing a trephination of approximately 80% stromal thickness, as measured by pachymetry. After removal of the anterior stoma, a needle is inserted into the residual stroma to inject air or viscoelastic to dissect Descemet’s membrane. These procedures are inherently difficult and intraoperative rates of Descemet’s membrane perforation between 4-39% have been reported. Optical coherence tomography (OCT) provides high-resolution images of tissue microstructures in the cornea, including Descemet’s membrane, and allows quantitation of corneal layer thicknesses. Here, we use crosssectional intraoperative OCT (iOCT) measurements of corneal thickness during surgery and a novel micrometeradjustable biopsy punch to precision-cut the stroma down to Descemet’s membrane. Our prototype cutting tool allows us to establish a dissection plane at the corneal endothelium interface, mitigates variability in cut-depths as a result of tremor, reduces procedure complexity, and reduces complication rates. iOCT-guided modified DALK procedures were performed on 47 cadaveric porcine eyes by non-experts and achieved a perforation rate of ~5% with a mean corneal dissection time <18 minutes. The procedure was also successful performed on a human donor eye without perforation. Our data shows the potential for iOCT-guided precision anterior segment surgery without variability as a result of tremor and improvements to standard clinical care.