The peptide hormone Insulin-like factor 3 (INSL3) is a marker for Leydig cell function and the clinical use of serum INSL3 measurements has been suggested by several groups.i) To establish a reference range for LC-MS/MS of serum 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 reference range based on LC-MS/MS of serum from 1073 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 (95% CI: 0.9 - 2.7) in young men (19 - 40 years) and decreased slightly in older men (0.1 µg/L per decade). Serum T, but not INSL3, was associated with body mass index or body fat percentage and with alcohol consumption. Smoking was positively associated with serum T, but negatively associated with INSL3. There were significant diurnal variations in both INSL3 and T in men (p < 0.001), but serum INSL3 varied substantially less, as compared to serum T (+/- 11% vs. +/- 26%). Mean serum INSL3 increased after hCG stimulation, but less than T (+ 17% vs. + 53%). In both healthy men and in patients suspected of testicular failure baseline serum INSL3 was closer associated to the hCG-induced increase in serum T, than baseline T itself.Measurement of serum INSL3 by LC-MS/MS has promise as a marker of testicular disorders.