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• Letrozole can be diverted from its therapeutic use. • Letrozole counteracts feminizing effects due to anabolic steroids abuse. • Hair is the specimen of choice to document long-term drug exposure. • Two independent methods (LC-MS/MS and LC-HRMS) are essential for legal rules. Letrozole is a reversible aromatase inhibitor, used in the treatment of hormone-dependent woman cancer. No indication for medical use is available for men. In recent years, several cases of doping with letrozole have been observed, especially among high level athletes. Aromatase inhibitors reverse the harmful effects (feminizing) of the abuse of anabolic androgenic steroids. Letrozole is included on the list of products prohibited in- and out-competition by the World Anti-Doping Agency, under section S4.1. The aim of the present work was to develop a specific method to identify letrozole in human hair of a male amateur athlete by LC-MS/MS and confirmation by LC-HRMS, after incubation of 20 mg of matrix in 1 mL of methanol. The chromatographic separation was performed using a reverse phase column HSS C18 with a gradient elution of 15 min (from 87% to 5% of formate buffer adjusted to pH 3). Linearity was observed from 1 to 1000 pg/mg (r2 = 0.9999), after spiking blank hair with the corresponding amounts of letrozole. The limit of detection was estimated at 0.5 pg/mg and the lower limit of quantification was the first point of the calibration curve, i.e. 1 pg/mg. The precision was lower than 20% and there was no interference with the analytes by chemicals or any extractable endogenous materials present in hair. Letrozole was identified in the male amateur athlete hair at 310 pg/mg (segment 0–2 cm) and 245 pg/mg (segment 2–4 cm). [ABSTRACT FROM AUTHOR]