Copyright © 2018 Chen and Nichols. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. The lipid composition of human meibomian gland secretions (meibum) has been analyzed using both targeted and untargeted mass spectrometric approaches, each of which has its advantages and disadvantages. Herein we report the results of shotgun lipidomic profiling of human meibum using a new approach that combines the advantages of targeted and untargeted analyses to yield highly sensitive and comprehensive profiles. Samples containing an estimated 7–13 µg (8–16 nL) of human meibum lipids were analyzed using MS/MS all , an untargeted approach for MS/MS. Using MS/MS all with ESI and successive polarity switching, we obtained tandem mass spectra in both modes at every 1 Da step for all ions in the m/z 200–1,200 range. In approximately 12 min, a total of 2 MS spectra and 2,000 MS/MS spectra were acquired for each sample, from which targeted analysis information was extracted. This approach allowed for the comprehensive and highly sensitive detection of meibum lipids, including species low in abundance. Altogether, more than 600 unique lipid molecular species were identified in meibum, 3 times more than previously reported in untargeted analyses of meibum samples. This untargeted MS and MS/MS all approach may be extended to other biological systems for the detection of lipids with sensitivity comparable to targeted analysis.