The search for proteomic biomarkers in ocular disease is one of the most important research directions in recent years. Reliable biomarkers can be an immense adjuvant for both diagnostic and therapeutic approaches. There is no more readily available ocular tissue for proteomic analysis than tear film, which makes an interesting target for the biomarker search. Tear film is a complex fluid consisting of a superficial lipid layer, which covers the aqueous-mucous layer. Its complexity makes it a perfect candidate for all the “omics” approaches. Glaucoma, cataract, age-related macular degeneration, and other diseases are commonly thought to have a multifactorial background. Currently, no reliable non-invasive tests are available that would help physicians with screening and further patient management. The aim of the study is to present modern methods of measuring biomarkers in tears, with particular emphasis on spectrometric methods, and to discuss their diagnostic and therapeutic usefulness.