Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry.