For poly(ethylene glycol)-derivatized liposomes (PEG-liposomes), the chemical stability of both the phospholipid ester groups and the urethane anchor between the PEG chain and the phospholipid are two important factors determining their long-term stability. High-performance liquid chromatography (HPLC) quantification of PEG-derivatized phospholipids such as PEG-distearoylphosphatidylethanolamine (PEG(DS)PE) is hampered by the molecular mass distribution of the PEG polymer. We describe a novel Fourier transform infrared (FTIR) method to monitor the hydrolysis of (PEG-)phospholipids. The integrated intensities of the C=O stretching bands of the various components of hydrolyzed (PEG-)phospholipid samples are determined by curve fitting. Separation of intact lipids and hydrolysis products is not required. To validate the method, the hydrolysis of dipalmitoylphosphatidylcholine/distearoylphosphatidylethanolami ne (DPPC/DSPE) liposomes was monitored both by HPLC and FTIR. The results from both methods are in excellent agreement. Under the experimental conditions (pH 9, 70 degreesC), the hydrolysis rate constant of esters in liposomes was hardly affected by the presence of hydrophilic PEG chains at the surface of the liposomes. The stability of the PEG-phospholipid linkage could also be assessed. Although the urethane group is probably located in a more hydrophilic environment than the ester groups, hydrolysis of the PEG-PE anchor is still much slower than that of the esters.