Background and Aim:Ophthalmological cytology is an easy, informative, rapid, and commonly-used low-cost diagnostic method, but sample collection and preparation are essential steps in obtaining qualitative material for cytological evaluation. This study aimed to evaluate cytological smear quality and animal discomfort after single or three serial conjunctival scrapings in normal cat eyes using five sampling methods.Materials and Methods:Five cytology methods (mini brush, cotton swab, soft brush, Kimura spatula, and cytobrush) were used in 50 eyes (10 with one scraping and 10 with three consecutive scrapings for a particular method) in complete 25 clinically and ophthalmologically healthy cats of different ages, sexes, and breeds. Ocular discomfort (1 = eyes open, 2 = partially open, and 3 = eyes squinted), average cell count (ten 10× fields), cell distribution (ten 100× fields: 0 = all cells are aggregated, 1 = <25% cells are evenly distributed, 2 = 25–50% cells are evenly distributed, and 3 = >50% cells are evenly distributed) and sample quality – aggregates (two cells and more), mucus, and artifacts (1+ = fair, 2+ = moderate, and 3+ = high amount) were evaluated.Results:The discomfort scores for the mini brush, cotton swab, soft brush, spatula, and cytobrush after a single and three scrapings were 1, 1, 1, 2, and 3, respectively. The average cell counts ± standard deviation after one and three scrapings were as follows: mini brush 11.15 ± 13.87 and 7.55 ± 12.7; cotton swab 7.17 ± 10.20 and 10.00 ± 16.44; soft brush 19.45 ± 22.22 and 8.55 ± 13.82; spatula 17.15 ± 32.94 and 13.85 ± 22.01; and cytobrush 13.35 ± 18.33 and 13.05 ± 19.29, respectively; the cell distributions were 3, 3, 3, 1, and 1 after single scraping and 3, 3, 2, 0, and 2 after three scrapings, respectively.Conclusion:The mini brush was the optimal method since it produced less discomfort, fewer artifacts, and the highest smear quality. Spatula smears were difficult to evaluate due to material thickness. The highest mucus and aggregate amounts were found in cytobrush, cotton swab, and soft brush samples. In this study, small number of samples per each sampling method is a major limitation.