Résumé -Mots clés : Lait, suivi biologique thérapeutique, benzodiazépines, HPLC Abstract -Objective: To validate an HPLC analytical assay of benzodiazepines in milk to assess the degree of exposure of the breastfed infant and set up a therapeutic drug monitoring. Methods: The linearity, the recovery, the within-day repetability and inter-day reproductibility over three days of diazepam, desmethyldiazepam, bromazepam, oxazepam and lorazepam were studied with three different matrices, cow's milk Candia , freeze-dried milk dedicated for infants O-Lac Enfamil and mother breast milk. Midazolam was used as internal standard. The chromatographic system consisted of a Nucleosil column with an UV detection (254 nm). The flow of the pump was adjusted at 1.2 mL/min with an acquisition period of 20 min. Results: The linearity for diazepam, bromazepam and lorazepam ranged from 50 to 5000 ng/mL whereas for desmethyldiazepam and oxazepam, it ranged from 100 to 10 000 ng/mL (r 2 > 0.99). The recovery was around 100% (± 15%) for each tested benzodiazepine. Within-day and inter-day coefficients of variation (CV) for the five benzodiazepines ranged from -15.3% to 17.4%. Conclusion: The validated method is simple, fast and applicable to maternal milk samples containing benzodiazepines.