The study investigates sex-based differences in the tear film proteomes of healthy male and female dogs using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF). The objective was to identify protein variations that could explain physiological differences and susceptibility to ocular diseases. Tear samples were collected from 22 dogs, and proteomic analysis revealed 446 common protein spots, with 8 exhibiting statistically significant differences between sexes. Seven proteins, including TIMP-2, PFK, and Annexin A13, were upregulated in females, while IL-33 was higher in males, indicating potential hormonal influences on tear film composition. These proteins are associated with tissue protection, metabolism, and immune responses, indicating potential hormonal influences on ocular surface homeostasis. The findings contribute to the understanding of tear film composition and may serve as a basis for further research into tear film biomarkers. This knowledge could support the development of non-invasive diagnostic approaches in veterinary ophthalmology.