Molar absorptivity of the infrared (IR) fundamental OH-stretching vibration band at 3550 cm−1 was determined for rhyolitic glasses. Five obsidian samples, unheated and heated at 500–700 °C using an internally heated pressure vessel, were used to evaluate the dependence of the molar absorptivity and final quenched H2O speciation on H2O contents and temperature. Water contents of the obsidians were measured by Karl-Fischer titration first, then the amount of unextracted H2O was calibrated by IR spectroscopy and a conventional vacuum extraction method. Total H2O contents of the obsidians were determined to be 0.24–1.25 wt%. IR spectra of the unheated and heated obsidian samples were obtained using an FT-IR microspectrometer. We determined the molar absorptivity for the 3550 cm−1 band to be 75 ± 4 L/mol/cm without significant dependence on the H2O contents and heating temperature. This value can be used to determine precise H2O contents up to 1.25 wt% in rhyolitic volcanic glasses.