In this article we describe the development of a microfluidic chip to determine the concentration of spermatozoa in semen, which is a main quality parameter for the fertility of a man. A microfluidic glass-glass chip is used, consisting of a microchannel with a planar electrode pair that allows the detection of spermatozoa passing the electrodes using electrical impedance measurements. Cells other than spermatozoa in semen also cause a change in impedance when passing the electrodes, interfering with the spermatozoa count. We demonstrate that the change in electrical impedance is related to the size of cells passing the electrodes, allowing to distinguish between spermatozoa and HL-60 cells suspended in washing medium. In the same way we are able to distinguish between polystyrene beads and spermatozoa. Thus, by adding a known concentration of polystyrene beads to a boar semen sample, the spermatozoa concentrations of seven mixtures are measured and show a good correlation with the actual concentration (R-2-value = 0.97). To our knowledge this is the first time that the concentration of spermatozoa has been determined on chip using electrical impedance measurements without a need to know the actual flow speed. The proposed method to determine the concentration can be easily applied to other cells. The described on-chip determination of the spermatozoa concentration is a first step towards a microfluidic system for a complete quality analysis of semen.