Only 2–5% of seminal fluid is composed of spermatozoa, while the rest is seminal plasma. The seminal plasma is a rich cocktail of organic and inorganic compounds including hormones, serving as a source of nutrients for sperm development and maturation, protecting them from infection and enabling them to overcome the immunological and chemical environment of the female reproductive tract. In this review, a survey of the hormones found in human seminal plasma, with particular emphasis on reproductive hormones is provided. Their participation in fertilization is discussed including their indispensable role in ovum fertilization. The origin of individual hormones found in seminal plasma is discussed, along with differences in the concentrations in seminal plasma and blood plasma. A part of review is devoted to methods of measurement, emphasising particular instances in which they differ from measurement in blood plasma. These methods include separation techniques, overcoming the matrix effect and current ways for end-point measurement, focusing on so called hyphenated techniques as a combination of chromatographic separation and mass spectrometry. Finally, the informative value of their determination as markers of male fertility disorders (impaired spermatogenesis, abnormal sperm parameters, varicocele) is discussed, along with instances where measuring their levels in seminal plasma is preferable to measurement of levels in blood plasma.