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The aim of this study was to develop a sensitive, reliable and high-throughput liquid chromatography – electrospray ionization – mass spectrometric (LC-ESI–MS/MS) method for the simultaneous quantitation of cortisol and cortisone in human saliva. Derivatization with 2-hydrazino-1-methylpyridine (HMP) was one of the most challenging aspects of the method development. The reagent was reacting with cortisol and cortisone at 60 °C within 1 h, giving mono- and bis-hydrazone derivatives. Investigation of derivatization reaction and sample preparation was detailed and discussed. Improvement of method sensitivity was achieved with charged derivatization and use of on-line solid phase extraction (on-line SPE). The lower limit of quantitation (LLOQ) was 5 and 10 pg/ml for cortisol and cortisone, respectively. The developed method was subsequently applied to clinical laboratory measurement of cortisol and cortisone in human saliva.Difficulties of the sample preparation and derivatization were solved during method development.On-line solid phase extraction was used due to the high sample volume injection.LC–MS/MS method with charged derivatization and on-line SPE was developed to quantify cortisol and cortisone in human saliva.