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Given that the peculiar redox behavior of ergothioneine involves a rapid regeneration process, the measurement of its precursor and redox metabolite hercynine could be particularly useful in assessing its role in oxidative stress or other biological processes. Thus, a LC-MS/MS method for the determination of hercynine concentrations in whole blood was developed. After lysis of red blood cells by cold water, samples were filtered on micro concentrators at a controlled temperature of 4 &#176;C. The clear filtered fluid was then treated with diethylpyrocarbonate to derivatize hercynine for the analysis by LC-MS/MS. The derivatized analyte was isocratically separated as a carbethoxy derivative on a C18 column with a mobile phase of an aqueous 0.1% <i>v</i>/<i>v</i> formic acid and acetonitrile (95:5). Effluents were monitored by MRM transitions at <i>m</i>/<i>z</i> 270.28&#8594;95 and 273.21&#8594;95 for hercynine and its deuterated counterpart, respectively. No cross-talk between MRM transitions was observed and a good linearity was found within a range of 35&#8315;1120 nmol/L. The LOD and LOQ were, respectively, 10.30 and 31.21 nmol/L with an intraday and intermediate precision below 7%. The average hercynine concentration in whole blood from 30 healthy male volunteers (aged 77 &#177; 12 years) was 178.5 &#177; 118.1 nmol/L. Overall, the method is easy to perform, allowing a rapid and accurate assessment of whole blood concentrations of hercynine.