Cytokines, a family of pleiotropic regulatory proteins, are critical mediators of both innate and adaptive immune responses. Growing evidence supports the involvement of cytokines in several physiological and pathological processes, including those related to reproduction. Semen contains a broad panel of cytokines, which play a key role in several reproductive processes, influencing both sperm function and modulating the uterine immune-cytokine network, essential for successful embryo development. In the last years, the assessment of a wide repertoire of seminal cytokines has been used as valuable biomarkers for several physio- and pathological conditions in mammalian species, including fertility disorders. In this chapter, we will describe several methods and protocols for the detection and/or quantification of cytokines in semen samples (seminal plasma and sperm cells), using the pig as an animal model. First, we will describe a protocol for the quantification of cytokine mRNA by real-time quantitative PCR and for in situ detection of cytokine production by immunohistochemistry in sperm cells. In addition, we will describe methods for quantification of cytokines in seminal plasma samples by enzyme-linked immunosorbent assay (ELISA) as well as by novel cytokine bead arrays (Luminex®).