Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author’s laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm motion characteristics using CASA, preparation of fluorochromes (SYBR-14/propidium iodide) utilized to distinguish between intact (i.e., viable or “live”) versus non-intact (i.e., nonviable or “dead”) sperm, and the analysis and interpretation of plasma membrane intactness using flow cytometry.