Flow cytometry is a powerful tool to characterize different cell traits, and has also demonstrated valuable for simultaneous analysis of various sperm quality traits such as plasma membrane and acrosome integrity. The objective of this study were to compare two different flow cytometers to reveal if there are differences between them. These two flow cytometers; Cell Lab QuantaTM (Beckman Coulter) and Beckman Coulter Epics XL (Beckman Coulter), use two different principles to calculate cell size, electric volume (EV) and forward scatter (FS), respectively. Egg yolk is a component that is often used in cryopreservation media to prevent cell damage by freezing. The presence of debris and non-sperm particles like egg yolk or bacteria can result in inaccurate results analyzing spermatozoa. FS in combination with side scatter (SS) signals which gives information about granularity of the cells has traditionally been used in combination to exclude non-sperm particles from the analysis. The challenge is to exclude particles with overlapping size and granularity to the cells that are analyzed. Traditional gating based on FS and SS plot, excludes much of these non-sperm or debris events, but still it may be challenging to discriminate events with both overlapping particle size and fluorescence signals from spermatozoa. The flow cytometry analysis can thereby give inaccurate results, and different approaches for the two instruments were investigated. The aim of the study was to test whether measurement of cell size by EV is better than FS to differentiate spermatozoa from other particles or debris in the flow cytometric assessment. We also tested if incubation of thawed semen affects the separation of sperm cells from debris particles.