Circulating levels of gonadotropins, sex steroids, binding globulin, and other hormones of the hypothalamus–pituitary–gonadal (HPG) axis are finely orchestrated to regulate testicular function. Hyper- or hyposecretion of some or multiple of these players defines different clinical conditions, requiring different therapeutic approaches. Therefore, the correct management of various forms of male reproductive/sexual disorders requires the reliable and comprehensive laboratory assessment of multiple hormones. Overall, hormones of the HPG axis present a large diversity in terms of chemical and physical nature, circulating levels, intra- and interindividual variability, stability in ex vivo conditions, and other aspects. Therefore, each hormone requires dedicated preanalytical and analytical methodologies and poses specific challenges that scientists are still actively addressing. Immunoassays are the dominating technique in clinical laboratories. They exhibit unsurpassed versatility in the measurement of analytes of various nature and dimensions and overall good sensitivity and specificity. However, several drawbacks mine the inter-laboratory reproducibility and the accuracy of individual determinations due to frequently occurring interferences. Nowadays, an alternative to immunoassays is available for small hormones such as sex steroids. Indeed, liquid chromatography–tandem mass spectrometry is definitively improving accuracy and sensitivity of steroid determinations, offering a promising possibility to achieve a final harmonization of measurement. In this chapter, the hormones involved in the physiology of HPG axis and analytical techniques for their determination will be discussed.