The assessment of mammalian fertility, and the agents that affect it, is of increasing concern in medicinal, environmental, and agricultural science. The viability, integrity, and overall state of the male gamete (sperm) is an essential factor that must be considered in such studies. Traditional potency evaluations tend to be labor intensive and often are not precise. A CE-LIF technique for determining the viability of boar sperm was developed using the fluorescent stains SYBER-14 and propidium iodide. The buffer type, pH, ionic strength, applied voltage, and polymer additive must be optimized in order to obtain sharp peaks and accurate results. Extender solutions that are widely used in artificial insemination programs were found to be compatible and even beneficial for these CE-LIF experiments. A single viability assay takes less than 10 min, which is significantly faster than most other procedures. The compaction or focusing of the sample zone seems to be similar to that reported previously for microorganisms in CE.