Assessment of sperm viability is an important component for evaluating stallion sperm quality. The flow cytometer is considered the standard in the assessment of sperm plasma membrane integrity (viability); however, this instrument is costly to purchase and use, and it requires an experienced technician to operate it. The growing practice of assisted reproductive technologies (ARTs) in the equine industry has increased the need for an accurate but cost-effective means of determining sperm membrane viability. The NucleoCounter® SP-100TM is reported to be an accurate, easy-to-perform, and an efficient stallion-side test for sperm membrane viability. To evaluate usefulness of the NucleoCounter® SP-100TM for assessing sperm membrane integrity, neat semen was subjected to four treatments with varying seminal plasma volumes and sperm concentrations. Sperm membrane viability was assessed immediately, and at 24 and 48 hours after cooled-storage using three methods: 1) flow cytometer utilizing the fluorescent vital stains SYBR-14/propidium iodide; 2) NucleoCounter® SP-100TM utilizing the fluorescent vital stain propidium iodide; 3) eosin-nigrosin stained air-dried smears of semen. Sperm motion characteristics (total and progressive motility) were assessed using a computer assisted sperm motion analyzer (CASMA) and results were compared to sperm membrane viability to determine the relationship between sperm membrane viability and motion characteristics. Results were compared statistically by: 1) analysis of variance (ANOVA); 2) linear regression analysis; 3) coefficient of variation on untransformed and transformed data (arc sine square root); and 3) the agreement of two instruments, by means of which the difference between measurements of the two instruments were plotted on the y-axis and the average of measurements from the two instruments were plotted on the x-axis. Results obtained with the NucleoCounter® SP-100TM agreed best with the flow cytometer, and least with eosin-nigrosin staining. Coefficients of variation were ≤ 5% for the three methods (transformed data). Sperm motion characteristics and sperm viability were similar among treatments at Time 0. At Times 24 and 48, sperm motion characteristics decreased at a more significant rate compared to viability in the treatments containing ≥ 50% seminal plasma, whereas differences among treatments were only significant at seminal plasma concentrations above 50% when only sperm membrane viability was considered.