AbstractThe objective of this study was to evaluate the accuracy of a novel, portable device (iSperm® Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm® Equine (Aidmics Biotechnology), Androvision® (Minitube) and NucleoCounter® SP‐100™ (ChemoMetec). The total motility and progressive motility were determined by the iSperm® Equine and the Androvision® using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above‐mentioned devices. There was a significant correlation between the concentrations measured with iSperm® and NucleoCounter® at all the measured dilutions. Moreover, <10% difference in concentrations was observed between the iSperm® and NucleoCounter® using the Bland–Altman test. There was also a significant correlation between iSperm® and Androvision® for total and progressive motility. In the second experiment, the parameters used in the Androvision® were modified to match those of the iSperm®. Total motility and progressive motility of frozen/thawed semen samples (n = 10) were determined, and the similarity between the Androvision® and iSperm® was confirmed by correlation studies and Bland–Altman test. The results of these experiments demonstrate that the iSperm® offers a reliable and practical alternative for the semi‐automated measurement of concentration and motility of stallion semen in the field. The iSperm® enables the practitioner to obtain objective and repeatable measurements on a variety of semen types (fresh, cooled and frozen) in the field at the time of insemination and thus acquire more insight into the quantity and quality of the provided insemination doses. This mare‐side diagnostic tool may help practitioners in identifying presumed subfertility problems more rapidly and act accordingly.