Purpose: The aim of this study was to evaluate the effect of corneal cross-linking on central endothelial cell density (ECD) by comparing masked measurements before and immediately after preparation of cross-linked (CXL) and non–cross-linked (non-CXL) donor corneas. Methods: Thirty donor corneas were randomized 1:1 to either CXL or non-CXL using a modified Dresden protocol. Specular microscopic images of the central corneal endothelium were obtained within 36 hours of recovery and within 3 hours of preparation completion. Images were sent to a central cornea image analysis reading center for ECD determination in a masked, dual-graded manner. Results: Before and after preparation, the mean ECD was 2567 ± 305 cells/mm2 and 2525± 339 cells/mm2 for the CXL group (n =15) and 2652 ± 365 cells/mm2 and 2572 ± 513 cells/mm2 for the non-CXL group (n =15), respectively. The mean difference in ECD before and after preparation was not statistically significant for the CXL and non-CXL groups (P = 0.36; P = 0.31, respectively). In addition, an independent sample t test showed no statistically significant difference in the ECD change between the CXL and non-CXL groups (P = 0.67). Conclusions: Our specular microscopic findings show that ex vivo cross-linking of donor corneas does not seem to produce acute decrease of central endothelial cell density. These findings are encouraging and assist in warranting a clinical trial examining its value in reducing refractive errors and improving postoperative visual outcomes in eyes with keratoconus that undergo keratoplasty.