AIM: To evaluate the quantitatively changes in lipid layer thickness (LLT) when 3% diquafosol eye drop is used for dry eye patients using the tear film interferometer. METHODS: A total 124 participants (32 males, 92 females; mean age, 28.9y) diagnosed with dry eye disease (DED) received topical instillation of 4 ophthalmic solutions in one eye: diquafosol, normal saline, 0.1% sodium hyaluronate and 0.3% gatifloxacin, in a masked manner. LLT was measured using an interferometer at baseline and 20min after the instillation of each ophthalmic solutions. RESULTS: Changes of LLT after instillation (nm, mean± standard error) were as follows: 12.6±2.0 for diquafosol (P<0.001), 1.2±2.2 for normal saline (P=0.301), 1.5±2.0 for hyaluronate (P=0.495), and 0.5±3.2 for gatifloxacin (P=0.884). CONCLUSION: Topical instillation of diquafosol increases tear film LLT in DED patients. Diquafosol 3% eye drop might be effective treatment option of evaporative DED with meibomian gland dysfunction.