Albrethsen J, Johannsen TH, Jørgensen N, et al. - In the present study, the researchers established a reference range for LC-MS/MS of serum Insulin-like factor 3 (INSL3) in healthy boys and men and ii) to compare the associations of serum INSL3 and testosterone (T) to pubertal stage, life style factors, diurnal variation, body composition and human chorionic gonadotropin (hCG) stimulation. In a LC-MS/MS serum-based reference sample of 1,073 boys and men, INSL3 increased from levels close to the detection limit (0.03 µg/L) in pre-pubertal boys to a maximum mean level of 1.3 µg/L in young men (19 – 40 years) and marginally decreased in older men (0.1 µg/L per decade). Serum T was correlated with BMI or body fat percentage and with alcohol intake, but not INSL3. Smoking was positively correlated with serum T, but negatively related to INSL3. For both healthy men and patients suspected of baseline serum INSL3 testicular failure, the hCG-induced serum T increase was more closely related than baseline T itself. The measurement of serum INSL3 by LC-MS/MS has been promised as a marker for testicular disorders.