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Clomiphene is a selective estrogen receptor modulator. It is indicated for the treatment of female infertility issues but in sport, it can be misused to stimulate endogenous testosterone secretion in men. Therefore, it has been prohibited at all times by the World Anti-doping Agency. The aim of this study was to get data to be able to interpret concentrations in athletes. A healthy volunteer (male, 62 years-old) ingested a single therapeutic dose of clomiphene (Clomid™, 50 mg). Strands of hair (blond, 4 cm) were collected one month after the ingestion. Body hair (beard, axillary, pubic and chest hair), and finger and toenails were collected over 4–5 months. A previous method was modified to identify and quantify clomiphene in keratinous matrices. 30 mg of specimen were sonicated and incubated in 1 mL of methanol, in presence of 200 pg of clomiphene-D 5 (internal standard). After centrifugation and evaporation of the organic phase, the samples were analyzed using LC-MS/MS. Linearity was verified in hair and nail clippings between 1 and 500 pg/mg. The limits of detection and quantification were determined at 0.3 and 1 pg/mg respectively. The study demonstrated that clomiphene tested positive in all the analyzed specimens at 9 pg/mg in head hair, from 28 to 486 pg/mg (body hair) and from 4 to 57 pg/mg (nails). Clomiphene was identified for the first time in multiple keratinous matrices. This study demonstrated that a single oral therapeutic dose is detectable in keratinous matrices over a long period of time. • A single dose of clomiphene (50 mg) is detectable in head hair, body hair and nail clippings. • A single therapeutic dose is detectable for a long period in keratinous matrices. • Higher concentrations of clomiphene are identified in body hair than in head hair. [ABSTRACT FROM AUTHOR]