In the basic clinical work-up of infertile couples, a semen analysis is mandatory and the sperm concentration is one of the most essential variables to be determined. Sperm concentration is usually assessed by manual counting using a haemocytometer and is hence labour intensive and may be subjected to investigator bias. Here we show that image cytometry can be used to accurately measure the sperm concentration of human semen samples with great ease and reproducibility. The impact of several factors (pipetting, mixing, round cell content, sperm concentration), which can influence the read-out as well as inter-operator and -cytometer variation on two different image cytometers (NC-3000 and SP-100) were evaluated. Furthermore, 725 semen samples were assessed both by manual assessment (WHO recommended method) and by image cytometry and tight correlations between the measured concentrations were shown. Moreover, by evaluation of repeated measurements it appeared that image cytometry produced more consistent and accurate measurements than manual counting of human spermatozoa concentration. In conclusion, image cytometry provides an appealing substitute of manual counting by providing reliable, robust and easy measurement of human sperm concentration.