Assessment of sperm vitality is an important component of semen analysis. It helps to distinguish spermatozoa that are alive and immotile from those that are dead. Sperm vitality can be assessed routinely on all semen samples by assessing the membrane integrity of the cell by identifying the spermatozoa with an intact cell membrane. This can be done by using 1) the dye exclusion test or 2) the hypotonic or hypoosmotic swelling test. Sperm vitality can therefore provide a good comparison with the motility of the sample. Eosin is used as a marker for dead cells because eosin can penetrate the cells when the membrane is damaged, while cells that have an intact membrane remain unstained. Nigrosin is a background stain that increases the contrast to the otherwise faintly stained cells [1, 2, 3, 4]. Both the single step and two-step staining using eosin and nigrosin have been used to assess sperm vitality. Both the wet preparation and the air-dried methods have been compared to study the correlation with motility [5, 6, 7]. The wet preparations evaluated by using either positive or negative phase-contrast microscopy consistently showed higher percentage of nonviable cells compared to the air-dried eosin-nigrosin smears. The air-dried smears have consistently shown that the sum of the motile (viable) and stained (presumed dead) preparations never exceeded 100 percent indicating that the air-dried method is the method of choice for determining vitality. In this chapter, we describe the staining protocols for vitality, the cut-off of motility when vitality must be tested, indications for poor motility and quality control recommended for performing sperm vitality in conjunction with basic semen analysis.