The tetraether index of tetraethers consisting of 86 carbons (TEX 86) is a novel proxy applied to obtain paleotemperature reconstructions from marine and lacustrine settings. It is usually applied alongside the branched vs isoprenoid tetraether (BIT) index, which provides paleoenvironmental information as well as information on the reliability of TEX86. Both indices are calculated via the analysis of glycerol dialkyl glycerol tetraethers or GDGTs by means of highperformance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/ APCI-MS). Here we test the performance of alternative methods for sample cleanup and instrumental analysis. In particular, we evaluate using alkaline hydrolysis as an alternative cleanup step to alumina column fractionation and show that the resulting TEX86 and BIT are statistically equivalent. We also test two different adsorbents in the activated or deactivated state for preparative column fractionation and show that any of them can be used to measure TEX86 but that a certain discrimination between GDGTs used in the BIT index can occur. Regarding the mass spectrometer design, an ion-trap is shown to be as precise as a quadrupole mass spectrometer for GDGT analysis. Some differences are observed for TEX86 and especially for BIT values obtained from both MS designs. We provide evidence that the APCI conditions are at least partly responsible for these differences. We recommend caution when comparing BIT values among laboratories as this index seems to be especially sensitive to analytical conditions. © 2009 American Chemical Society.