Meibomian glands (MG) secrete an oily substance, meibum, that spreads across the ocular surface and mixes with secretions produced by other ocular structures to create a thin film. The protective efficacy of the tear film is believed to be related to the chemical composition of the lipid layer. We reviewed the literature describing the composition of human MG secretions and have provided an overview on methods of collecting meibum samples, methods of lipid analyses, and the results obtained in previous studies. The usefulness and quality of the data obtained about meibum depend on proper sampling and the analytical techniques used. Historically, several methods have been developed, which have yielded contradictory data regarding meibum sample collection and analytical techniques. Based on review of the literature, the major lipids present in meibum are of nonpolar origin: waxes, sterols, and sterol esters, followed by triacylglycerides and fatty acids. The amphiphilic lipids, diacylglycerides, were reported in fewer studies, and monoacylglycerides were reported in only two. Information on the composition of the polar lipids is more controversial. Meibum phospholipids were found in small amounts (16% or less) in some studies, but not in others. Thus, meibum is a complex mixture of lipid molecules. Historical analytical inconsistencies may be partly explained by limitations of past analytical procedures and by the consideration that the tear film lipid layer may have contributing sources other than meibum.