Semen is one of a few naturally occurring monocellular suspensions, and therefore sperm function analysis by flow cytometry and utilization of fluorochromes is an ideal technique for high throughput, multiparametric, and accurate analysis. Sperm quality assessment often includes measures of cellular integrity and potential capability of spermatozoa to fertilize and stages of capacitation. When deciding on and setting up a sperm assay using flow cytometry, there are several factors to consider which affect the assay and the measured output. These include but are not limited to instrument type and settings and sample preparation. The classic sperm assays which use flow cytometry are sperm viability, acrosomal integrity, membrane stability, and mitochondrial status. Here, we describe principles and procedures to set up and develop these sperm function analyses using traditional flow cytometry.