Abstract The objective of this study was to evaluate the repeatability and accuracy of canine sperm motility (total and progressive) assessment with a tablet-based Canine iSperm instrument compared to computer-assisted sperm analysis (CASA). The experiment used fresh and frozen/thawed canine semen samples for comparisons of semen analysis parameters (concentration, total motility, and progressive motility) between a CASA system, iSperm, and NucleoCounter SP-100 (concentration) instruments. Spearman’s Rho correlational analysis was used to identify significant associations between motility assessment methods. Significant positive correlations were found between CASA assessment and iSperm for both progressive and total motility measurements. We also determined the coefficient of variation (CV) for repeatability of sample analysis for iSperm and CASA for fresh sperm, wherein each sample was assessed 10 times on both devices. For fresh and frozen-thawed samples, concentration assessment by iSperm showed high variability (CV= 19.9 ± 1.5%). For iSperm assessment of total and progressive motility, the CVs were 6.3 ± 0.5% and 10.7 ± 0.8%, respectively. The results indicate that the iSperm application offers an accurate and alternative measurement of motility to traditional CASA analysis, though caution should be taken when assessing concentration due to the high CV observed in this study.