In normal clinical practice, testicular evaluation in boys has relied on palpation and testosterone determination after hCG stimulation, which reflects the activity of interstitial Leydig cells. However, the most active compartment of the testis before puberty is the seminiferous tubule compartment, in which Sertoli cells proliferate and secrete anti-Müllerian hormone (AMH) and inhibin B. The recent development of commercially available assays for these two peptides has provided the pediatrician with excellent tools to assess the existence of functional testicular tissue in boys with no need for hCG stimulation. Serum AMH determination is also useful to assess testicular tissue mass and function in patients with intersex disorders. The determination of testosterone, its precursors and dihydrotestosterone, after hCG stimulation, should be reserved for situations in which Leydig cell function needs to be specifically assessed.