Flow cytometry allows the assessment of multiple sperm parameters following a diverse number of protocols. Here, we describe three methods for evaluating critical aspects of sperm quality: the double stain SYBR-14 and propidium iodide (PI) for assessing sperm viability; the double stain PNA-FITC (peanut agglutinin-fluorescein isothiocyanate) and PI for assessing acrosomal integrity combined with sperm viability; and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide) for assessing mitochondrial membrane potential. These three stains are widely used and can be analyzed with basic flow cytometers.