We optimized a mercury (Hg) speciation extraction method for human hair in combination with species-specific isotope-dilution analysis by gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS). The method was validated on human hair reference material RM (IAEA-086), which is recommended for analysis of monomethylmercury (MMHg) and inorganic mercury (IHg). Three reagents, hydrochloric acid (HCl), nitric acid (HNO3), and tetramethylammonium hydroxide (TMAH), and three extraction procedures, at ambient temperature for 12 h, microwave-assisted at 75 °C for 6 min, and oven heated at 80 °C for 2 h were tested. Extraction efficiency, recovery, and potential species transformations were evaluated for each method. The most efficient procedures, with recovery of ~90 % for each species with limited demethylation (<5 %) and methylation (0 %), were HNO3 digestion, irrespective of temperature, and microwave-assisted TMAH extraction. Acidic extraction with HCl induces significant demethylation, with production of artifacts. To correct for potential demethylation artifacts we recommend spiking with isotopically enriched standards before the extraction step.