The tear film of the typical land mammal is generally comprised of three layers--the mucin-rich innermost layer, the aqueous layer, and the outer lipid layer at the air interface. Our laboratory's histological evidence to date has shown a lack of evidence of meibomian glands in cetaceans thus a predicted lack of meibum secretion that forms the tear film lipid layer. However, the tear film layer of the Orca whale is clinically very viscous compared to that of a land mammal, suggesting that the mucin layer may predominate. There is concern as to whether topical therapy with medications can penetrate the thick viscous orca tears and enter the cornea or anterior chamber. Thus, the purpose of this work was to evaluate the cetacean tear film using mass spectrometry based methods. A 1mL tear film sample from an Orca whale was collected, stored in dry ice, and transported for analysis. The sample was analyzed by 1D SDS-PAGE and visualized with Coommassie staining. After staining, an entire gel lane was divided into 40 small sections and subject to in-gel trypsin digestion. Peptides were extracted from the bands after digestion and analyzed by LC-MS/MS. Sequence information from the MS/MS data was processed using MASCOT. Proteins identified in Orca tear film included lysozyme, submaxillary apomucin, immunoglobulin V(H), immunoglobulin J chain, immunoglobulin A heavy chain, MUC19, protein similar to mucin 19, immunoglobulin M heavy chain, solute carrier family 25 member 36, albumin, complement C3 precursor. This is notably different from what is commonly observed in human tear film where over 100 proteins have been identified, and the five most frequently observed proteins including lactoferrin, lipocalin 1, lysozyme, lacritin and a variety of immunoglobins. Also of note is the high amounts of mucins observed in the orca tear which are not observed at the same high level in human tears. In humans, the goblet-cell derived preocular mucins function to fill in any irregularities of the corneal surface and the mucoproteins trap bacteria and foreign particles thus protecting the ocular surface. In cetaceans, the mucin layer appears to be much more substantial, and may function as a barrier at the eye/water interface. This may have clinical implications relative to the use of topical medications ability penetrate the mucins. One approach would be the addition of mucolytics (e.g., acetyl-cysteine) to existing medications to improve the effectiveness of topical medications for these animals. Alternatively, oral medications may be a more effective way to treat ophthalmic problems of cetacean.