Male reproduction, sexual differentiation, maintenance of sexual characteristics and ageing are all under strict control of the hypothalamic–pituitary–testis axis. A hypothalamic pulse generator releases gonadotropin-releasing hormone into the hypothalamic–pituitary portal system. In response to this stimulus, the anterior pituitary secretes follicle-stimulating hormone (FSH) and luteinizing hormone (LH) which, in turn, stimulate gonadal activity. Through the peripheral effects of these hormones, spermatogenesis, testosterone (T) production and overall male health are ensured. Any disturbance of this pathway leads to gonadal dysfunction, decreased testosterone production and different clinical manifestations, depending from the time of onset. In this context, androgen deficiency can be the result of defects in testosterone production and spermatogenesis, associated genetic factors and/or history of androgen therapy. These features comprise the challenge to have different diagnostic workups among males with paediatric, pubertal and late-onset hypogonadism (LOH). Acquired or genetic conditions at young age mainly cause delayed puberty. On the contrary, different causes lead to late-onset hypogonadism with signs and symptoms of androgen deficiency. The current chapter aims to provide an overview about testosterone physiology with a detailed and up-to-date description of diagnostic workups for paediatric, pubertal and late-onset hypogonadism.