Context Delayed puberty is a frequent complaint in males. The differential diagnosis between self-limited delayed puberty (SLDP) and congenital hypogonadotropic hypogonadism (CHH) is challenging. Commonly used endocrine tests, focusing on stimulated levels of LH or testosterone, are not satisfactory in making a diagnosis. Because FSH action on Sertoli cells results in testis enlargement and anti-M & uuml;llerian hormone (AMH) and inhibin B increased secretion, and the FSH-Sertoli cell axis function is detectable during normal childhood and early puberty, we tested whether the assessment of serum FSH, AMH, and inhibin B would be informative to distinguish between SLDP and CHH. Design We performed a prospective, nested case-control study in a cohort of male adolescents presenting with delayed puberty, comparing baseline serum reproductive hormone levels to identify predictive biomarkers of CHH, after having followed all participants prospectively until a final diagnosis was ascertained based on gold-standard criteria (age 18 years or >= 4 years after testis volume reached 4 mL). Results Of 65 participants who completed follow-up, 33 had a final diagnosis of SLDP and 32 of CHH. Serum FSH, AMH, and inhibin B showed better diagnostic efficiency than LH and testosterone for these differential diagnoses. FSH (IU/L)xinhibin B (ng/mL) < 92 and FSH (IU/L)xAMH (pmol/L) < 537 showed high sensitivity (>93%), specificity (>= 92%), predictive values (>92%), and positive likelihood ratio (>12) for CHH. The diagnostic performance remained 89.7% and 88.2% for FSH x inhibin B and FSH x AMH, respectively, when analyzed in patients without red flags (micropenis, cryptorchidism, and/or microorchidism). Conclusion Serum FSH combined with inhibin B or AMH is highly predictive to accurately distinguish between SLDP and CHH in adolescent males.