© 2019 Elsevier Inc. Purpose: The purpose of this work was to optimize methodology to analyze the human tear film lipids by using untargeted, direct infusion electrospray ionization-mass spectrometry (ESI-MS) to establish the analytical approach for a large-scale clinical translational study of tear film lipids in ocular surface disease, particularly associated with the O-acyl-omega hydroxy fatty acids (OAHFAs). Methods: Meibum and tear samples were collected from both eyes of five normal subjects without ocular disease using two different microcapillary collection tubes, glass and polytetrafluoroethylene (PTFE). An untargeted lipidomics approach was used to analyze the lipids in human tear and meibum samples using direct infusion ESI-MS in positive and negative ion modes. Direct and indirect quantification methods were evaluated. Results: The amount of OAHFAs measured in tears using these techniques was approximately 0.7–0.8% of the total lipids. More phospholipids, including phosphatidylcholine and sphingomyelin, were detected in the tear samples associated with glass microcapillaries compared to PTFE. Conclusions: Reliable assessment of lipids in small volumes of tear film is possible using high resolution, untargeted ESI-MS in positive and negative ion modes. Using this technique, the concentration of OAHFAs can be quantified, as can the presence of other polar lipids.