PURPOSE: To qualitatively assess the endothelial damage on Descemet-stripping automated endothelial keratoplasty (DSAEK) donor tissue resulting from a gentian violet marking pen. METHODS: An in vitro model was used, by using corneoscleral rims, DSAEK quality corneal donor tissue, and a gentian violet marking pen. After making a mark on the stromal side of a microkeratome-prepared DSAEK corneal button to confirm appropriate orientation of the donor tissue after insertion into the anterior chamber, the corneal tissue was returned to Optisol GS solution for 1 hour. A vital dye assay was used to identify devitalized and necrotic endothelial cells with alizarin red S and trypan blue. RESULTS: Corneal donor tissue evaluated with the gentian violet marking pen showed positive trypan blue staining, limited to the area marked with the gentian violet ink. CONCLUSIONS: Marking the DSAEK donor stromal surface with a gentian violet marker damages the endothelium. Surgeons should limit the size of the mark or use an insertion technique that avoids confusion about orientation of the donor cornea.