AbstractThe entire scalp hair of a self‐declared Kratom consumer of 3 grams per day was acquired during an ethical committee approved study. As no values of the concentration in hair of the two Kratom alkaloids mitragynine or 7‐hydroxymitragynine were found in the literature, an already established method for the analysis of benzodiazepines/z‐substances was extended for the detection of mitragynine and 7‐hydroxymitragynine with LC–MS/MS, and successfully validated. The limits of detection and quantification for mitragynine were 2 pg/mg and 4 pg/mg, respectively. Those of 7‐hydroxymitragynine were 20 pg/mg and 30 pg/mg, respectively. The method was applied to the entire scalp hair, divided in 91 regions, of the study participant. A narrow mitragynine concentration distribution with values between 1054 pg/mg and 2244 ng/mg (mean 1517 ng/mg) and no clear scalp region associated distribution pattern was obtained. 7‐Hydroxymitragynine was not detected in any hair sample. After validation, the method was established as routine and subsequently 300 samples (mainly abstinence controls for drugs of abuse) were analyzed, allowing the investigation of the prevalence of Kratom consumption in our population. None of the analyzed routine hair samples were positive for mitragynine or 7‐hydroxymitragynine, providing no evidence that Kratom consumption is prevalent in the investigated population.