Meibum is believed to be the major source of tear !lm lipids, which are vital in the prevention of excessevaporation of the aqueous phase. The complete lipid composition of meibum has yet to be established.While earlier studies reported the presence of phospholipids in human meibum, recent mass spectrometricstudies have not detected them. In this study we use electrospray ionisation tandem massspectrometry to investigate the presence of phospholipids in meibum and provide comparison to thephospholipid pro!le of tears.Lipids were extracted from human meibum and tear samples using standard biphasic methods andanalysed by nano-electrospray ionisation tandem mass spectrometry using targeted ion scans. A total of35 choline-containing phospholipids were identi!ed in meibum and the pro!le of these was similar tothat observed in tears, suggesting tear lipids are derived from meibum. The results shown here highlightthe need for a combination of optimised techniques to enable the identi!cation of the large range of lipidclasses in meibum.