PURPOSE. The purpose of this investigation was to characterize differentially expressed lipids in meibum samples from patients with dry eye disease (DED) in order to better understand the underlying pathologic mechanisms. METHODS. Meibum samples were collected from postmenopausal women with DED (PW-DED; n ¼ 5) and a control group of postmenopausal women without DED (n ¼ 4). Lipid profiles were analyzed by direct infusion full-scan electrospray ionization mass spectrometry (ESI-MS). An initial analysis of 145 representative peaks from four classes of lipids in PW-DED samples revealed that additional manual corrections for peak overlap and isotopes only slightly affected the statistical analysis. Therefore, analysis of uncorrected data, which can be applied to a greater number of peaks, was used to compare more than 500 lipid peaks common to PW-DED and control samples. Statistical analysis of peak intensities identified several lipid species that differed significantly between the two groups. Data from contact lens wearers with DED (CL-DED; n ¼ 5) were also analyzed. RESULTS. Many species of the two types of diesters (DE) and very long chain wax esters (WE) were decreased by ~20% in PW-DED, whereas levels of triacylglycerols were increased by an average of 39% ± 3% in meibum from PW-DED compared to that in the control group. Approximately the same reduction (20%) of similar DE and WE was observed for CL-DED. CONCLUSIONS. Statistical analysis of peak intensities from direct infusion ESI-MS results identified differentially expressed lipids in meibum from dry eye patients. Further studies are warranted to support these findings.