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Fertility of a male is sometimes difficult to assess and results obtained using regular sperm analysis methods are often inconclusive. Flow cytometry was proven to generate crucial information, that allows specialists to conclude upon the reproductive capacity of an individual. Together with computer assisted sperm analysis CASA systems, flow cytometers allow an in-depth analysis of fresh, chilled or frozen/thawed semen, which is currently essential for research purposes, but also for diagnosis of various male-related fertility disorders, both in veterinary and human medicine. Several parameters can be assessed using this method, such as sperm viability, acrosome integrity, mitochondrial activity, concentration and total sperm number, DNA fragmentation, capacitation, oxidative stress, etc. This paper provides a rapid reference to specialists involved in semen analysis by flow cytometry, regarding semen sample preparation, instrument setup, and evaluation of the most important sperm parameters (viability, acrosome reaction and mitochondrial activity).