Proteomic analysis of tear fluid is challenging mainly due to a wide dynamic range of proteins and small sample volume. However, recent advancements in mass spectrometry have revolutionized the field of proteomics through its unprecedented depth, speed, and accuracy even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass-spectrometry workflows for proteomic analysis of human tear fluid.