Although both serum inhibin B (Inh-B) and anti-Müllerian hormone (AMH) concentrations have been proposed as markers of spermatogenesis in men with subfertility, there are wide overlaps with fertile controls. The main aim of this study was to evaluate stimulated serum Inh-B and AMH concentrations in men with non-obstructive azoospermia (NOA). Thirty-seven men with NOA confirmed by testicular fine-needle aspiration and 17 fertile controls participated at this prospective, case-control study. All subjects underwent the Exogenous FSH SErtoli Reserve Test (EFSERT): estimation of serum Inh-B and AMH concentrations before, 24 and 48 hours after administration of 300 IU human recombinant FSH (hrFSH). Basal serum Inh-B and AMH concentrations, as well as Inh-B concentrations at 24 and 48 h were lower in men with NOA as compared to controls. No changes in Inh-B or AMH concentrations were recorded throughout the EFSERT in either men with NOA or controls nor when men with NOA were classified according to their clinical, hormonal and cytological diagnosis. Thus, stimulated serum concentrations of Inh-B and AMH, as obtained by an EFSERT, do not contribute to the diagnostic evaluation of the men with NOA, as the same information can be acquired by the basal serum concentrations of these hormones.