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To describe our process for preoperative screening and donor selection for ocular surface stem cell transplantation (OSST).A 7-year retrospective chart review was performed on limbal stem cell deficiency patients. The inclusion criterion was all patients who underwent an OSST procedure. The exclusion criterion was eyes with unilateral disease in which an autograft was performed. Data for human leukocyte antigen (HLA) typing, virtual crossmatching, donor-specific antibody, and panel reactive antibody level were obtained.Of the included 142 eyes (104 patients), 19 patients had no recorded living donor availability data, and HLA typing was not performed on 16 patients. A total of 94 donors (mean 1.4 donors/patient, range 1–6) were tested for 67 recipients. For 2 patients with graft-versus-host disease, no further HLA typing was needed, as the donors were known HLA-identical donors. For 47 patients, only 1 donor was tested, whereas multiple donors underwent HLA typing for 20 patients. There were 73 ABO (blood group)-compatible matches for the 61 tested recipients, and only 1 recipient did not have any ABO-compatible donor. For the virtual crossmatch, there were 5 patients who did not have a compatible donor (positive virtual crossmatch). The best available donor match was a sibling for 41 recipients (65%), a parent for 19 recipients (30%), and an offspring for 3 recipients (5%).Our protocol for OSST preoperative screening and donor selection minimizes the antigenic burden for transplanted tissue by selecting the best available donor match.