© 1994 SPIE. All rights reserved. A method has been developed to determine the exact fractional composition in binary mixtures of phospholipids at the air-water (A/W) interface by ATR-IR spectroscopy in combination with 31P NMR spectroscopy and Langmuir-Blodgett surface chemistry. The procedure utilizes the wavenumber shift observed upon the synthetic replacement of hydrogen with deuterium in the lipid acyl chains to separate the vibrational bands due to each component in mixtures of deuterated and normally protiated lipids. The C-H and C-D integrated areas obtained from the IR spectra of the binary mixture are correlated with the exact mole fractions as determined using high resolution 31P NMR spectroscopy. We have used this technique to measure the fractional composition of binary mixtures of phospholipids at the A/W interface continaining acyl chain perdeutereated phosphocholine lipids in combination with several protiated phosphoglycerol lipids.