Human tear is a biological fluid rich in lipids that is increasingly collected in clinical and biological research. The repertoire of small lipids or lipid mediators (often termed eicosanoids or oxylipins) found in human tear provides insight into metabolism of fatty acids and physiology of the ocular surface and Meibomian glands. Disturbances in the tear lipid mediators profile also occur during inflammation of the ocular surface that is not directly linked to lipid metabolism. The changes in the levels of pro-inflammatory and pro-resolution lipid mediators in the tear help assess the severity and stage of inflammation in ocular surface tissues. Mass spectrometry, used in the evaluation of tear lipid mediators, is an emerging tool in clinical diagnostics and personalized medicine. Here we describe the reproducibility, accuracy, and precision of quantifying lipid mediators in human tears, with a suggested method for tear collection and sample handling. The ranges of lipid mediators concentrations in tear fluid of healthy and diseased individuals with Meibomian gland dysfunction are reported, as well as the impact of age and disease on individual lipid mediators. We would like to recommend a set of guidelines, which can be further discussed in workshops. This will facilitate harmonization of future tear lipid mediators data across different instrument platforms in various laboratories. We hope that other fields requiring lipid mediators assays will also benefit from such an effort.Copyright © 2020. Published by Elsevier Inc.