PurposeTo determine the reliability of corneal endothelial cell density (ECD) obtained by automated specular microscopy versus that of validated manual methods and factors that predict such reliability.MethodsSharp central images from 94 control and 106 glaucomatous eyes were captured with Konan specular microscope NSP-9900. All images were analyzed by trained graders using Konan CellChek Software, employing the fully- and semi-automated methods as well as Center Method. Images with low cell count (input cells number <100) and/or guttata were compared with the Center and Flex-Center Methods. ECDs were compared and absolute error was used to assess variation. The effect on ECD of age, cell count, cell size, and cell size variation was evaluated.ResultsNo significant difference was observed between the Center and Flex-Center Methods in corneas with guttata (p = 0.48) or low ECD (p = 0.11). No difference (p = 0.32) was observed in ECD of normal controls <40 yrs old between the fully-automated method and manual Center Method. However, in older controls and glaucomatous eyes, ECD was overestimated by the fully-automated method (p = 0.034) and semi-automated method (p = 0.025) as compared to manual method.ConclusionOur findings show that automated analysis significantly overestimates ECD in the eyes with high polymegathism and/or large cell size, compared to the manual method. Therefore, we discourage reliance upon the fully-automated method alone to perform specular microscopy analysis, particularly if an accurate ECD value is imperative.