Due to its versatile nature and its corresponding anabolic and anticatabolic properties, insulin has been prohibited in sports since 1999. Numerous studies concerning its impact on glycogen formation, protein biosynthesis, and inhibition of protein breakdown have illustrated its importance for healthy humans and diabetics as well as elite athletes. Various reports described the misuse of insulin to improve performance and muscle strength, and synthetic analogs were the subject of several studies describing the beneficial effects of biotechnologically modified insulins. Rapid- or long-acting insulins were developed to enhance the injection-to-onset profile as well as the controllability of administered insulin, where the slightest alterations in primary amino acid sequences allowed the inhibition of noncovalent aggregation of insulin monomers (rapid-acting analogs) or promoted microprecipitation of insulin variants upon subcutaneous application (long-acting analogs). Information on the metabolic fate and renal elimination of insulins has been rather limited, and detection assays for doping control purposes were primarily established using the intact compounds as target analytes in plasma and urine specimens. However, recent studies revealed the presence of urinary metabolites that have been implemented in confirmation methods of sports drug testing procedures. So far, no screening tool is available providing fast and reliable information on possible insulin misuse; only sophisticated procedures including immunoaffinity purification followed by liquid chromatography and tandem mass spectrometry have enabled the unambiguous detection of synthetic insulins in doping control blood or urine samples.