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Purpose Pterygium is a common ocular surface disorder. Conjunctival autografting (CAG) following pterygium resection is the gold standard treatment. CAGs without Tenon's tissue provide better results but are more technically difficult to achieve. In this study, we evaluated the feasibility and reproducibility of femtosecond laser (FSL)-assisted CAG preparation. Methods Fifteen porcine globes were fixed in a suction holder and CAGs of different diameters were created by 1) an experienced consultant and 2) a less experienced fellow using the Ziemer LDV Z8. The CAG's dimension was measured and thickness analyzed by optical coherence tomography (OCT) and histology (HE). Statistical analysis was performed by Mann-Whitney-U, Wilcoxon and Spearman-test. Results FSL-assisted CAGs prepared at 100 μm (146.4 ± 45.7 μm) showed a significantly higher deviation from desired depth ( p = 0.04) and a higher variability ( p = 0.03) in thickness than those prepared at 60 μm (71.4 ± 12.7 μm). The experienced (68.3 ± 14.3 μm) and inexperienced surgeon (73.9 ± 11.9 μm) produced 60 μm grafts of comparable thickness ( p = 0.6) and variability ( p = 0.7). The CAG area measured after dissection (37.5 ± 12.1 mm 2 ) did not differ significantly from the FSL settings (40.6 ± 12.7 mm 2 , p = 0.3). FSL cutting time at 60 μm took 18.1 ± 2.2 s, at 100 μm 20.7 ± 2.4 s. Graft separation time was not significantly influenced by depth or surgeon. No buttonholes or CAG tags occurred during surgery. Conclusions The FSL allowed the accurate and reliable preparation of very thin CAGs, independent of surgeon experience and may represent a valuable tool in pterygium surgery.