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A method combining normal phase high performance liquid chromatography (HPLC) with positive ion atmospheric pressure chemical ionization mass spectrometry (APCI‐MS) was developed for the analysis of intact glycerol dialkyl glycerol tetraethers (GDGTs) in archaeal cell material and sediments. All GDGTs previously reported to occur in the thermophilic archaeon Sulfolobus solfataricus could be identified based on their mass spectra and retention time. Positive ion mass spectra consisted of abundant protonated molecules and fragment ions corresponding to loss of water and the glycerol moiety. In addition, two novel GDGTs representing alternative combinations of biphytanyl moieties were observed. Using this method, the tetraethers present in the thermophilic archaeon Metallosphaera sedula and two sediment samples were characterized. This rapid method will greatly contribute to the establishment of …