Biomarker based sperm quality evaluation adds value to conventional semen analysis for the treatment of human male infertility and amelioration of male reproductive performance in livestock species. The negative biomarker-based approach to andrological evaluation focuses on proteins and lectin ligands that are detectable predominantly or exclusively in defective spermatozoa. Contrary to conventional light microscopic semen analysis, the negative biomarker approach detects sperm defects at the molecular level, regardless of whether or not they are manifested in a visible morphological phenotype or diminished sperm motility. Such analysis lends itself to automated, objective testing by flow cytometry (FC), correlates with field artificial insemination (AI) fertility in livestock and reflects the outcomes of assisted reproductive therapies (ART) in humans. A prime example of a negative sperm biomarker in humans is the spermatid specific thioredoxin 3 (SPTRX3/TXDC8), present in testicular round spermatids of most mammalian species studied, but retained exclusively in the nuclear vacuoles and superfluous midpiece cytoplasm of defective human spermatozoa. Infertile couples in which men displayed elevated semen content of SPTRX3 had a significantly reduced chance of conceiving by ART and were more likely to suffer of recurrent miscarriage, while couples with low SPTRX3 were more prone to multiple births after multiple embryo transfer. Ubiquitin is a proteolysis-promoting posttranslational protein modifier that binds covalently to protein molecules destined for recycling by the 26S proteasome and is found on the surface of defective spermatozoa after sperm passage through epididymis. Elevated semen ubiquitin content correlates with fertility outcomes and conventional semen parameters in humans and animals, and can be measure by FC using anti-ubiquitin antibodies and fluorochromes with affinity to aggresomes, the cellular stress-induced aggregates of non-recycled ubiquitinated proteins. Lectin LCA (Lens culinaris agglutinin) binds to acrosomal surfaces of phenotypically normal spermatozoa but adheres to the entire sperm head and tail surface of defective spermatozoa. Lectin PNA (Arachis hypogaea/peanut agglutinin) has been known to bind exclusively to the acrosomal membranes/matrix of spermatozoa with malformed or damaged acrosomes, a property that has recently been utilized by our group to develop nanoparticle based bull semen purification method validated by field AI trials. In addition to biomarkers prevalent in defective spermatozoa, proteins important for normal sperm function may either be undetectable or over-expressed/ectopically localized in defective spermatozoa. The post-acrosomal sheath WW-domain binding protein PAWP has been implicated in the induction of oocyte activation during fertilization in humans and other mammals. Proper post-acrosomal sheath localization and FC-measured relative quantity of PAWP correlates with ART outcomes in infertile couples and with field AI fertility in livestock species. In AI bulls, semen PAWP content appeared predictive of sires’ fertility in a multiplex flow cytometric test also including probing of ubiquitin, aggresomes and lectin PNA binding sites. Altogether, the biomarker based technologies allow for unbiased diagnosis of human male infertility and informed clinical treatment decision making. In livestock reproduction, flow cytometric analysis has predictive value for future fertility of young sires. Semen nanopurification can be used to lower the sperm number per AI semen dose, allowing for increased production of semen doses per collection from sires with valuable genomes. These advances go hand-in-hand with innovation of flow cytometric technology such as the introduction of dedicated bench-top sperm flow cytometers and flow cytometers with single cell imaging capabilities, eliminating the need for extrapolation between biomarker quantification and light-microscopic analysis of sperm phenotypes. Supported by grants from USDA-NIFA, NIH-NICHD and Missouri Life Science Research Board, and by seed funding from the F21C Program, University of Missouri.