The iSperm® is a portable device for semen analysis. This study aimed to investigate its correlation with a conventional computer-assisted sperm analyzer (ISAS®v1) for the assessment of semen concentration and kinematic parameters in dogs (n = 224). The intra-assay variability of both devices and their ability to estimate semen concentration at a fixed value of 40 × 106/mL were also investigated. Results showed that the intra-assay variability was lower for the ISAS®v1 for all parameters compared to the iSperm®. Hence, iSperm® estimates were more variable in-between fields. Both the iSperm® and the ISAS®v1 were not reliable in estimating semen concentration (ISAS®v1: median 30 × 106/mL, interquartile range (IQR) 12, p < 0.01; iSperm®: median 35.12 × 106/mL, IQR 11.11, p < 0.01). Finally, positive correlations were found between both devices with stronger correlations obtained when four fields were analyzed by the iSperm®. However, the low number of spermatozoa analyzed per field and the inability to avoid artifacts are downsides that currently limit the reliability of the iSperm®. Therefore, the software of iSperm® needs some improvement to make it a valid and practical alternative to automated computerized systems for the analysis of canine semen.