In this study, a comprehensive comparative analysis of different evaluation methods of Elispot plates was performed. Three investigators using three different evaluation approaches read 50 randomly selected wells at three different time points. The methods were: (1) manual evaluation using a stereomicroscope, (2) automated evaluation using an image analysis reader system with reading parameters established by each investigator, and (3) automated evaluation using a reader system with preset reading parameters using assay-specific controls. We demonstrate that manual evaluation had the highest variability both within the same method and when comparing all methods, followed by automated evaluation with investigator-dependent parameters. The variability was low only when all investigators used the same parameters established using assay-specific controls. This variability was independent of operator or spot number per well. Based on this study, recommendations for standardization and validation procedures of Elispot assay performance and evaluation procedures are presented.