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Tear film hyperosmolarity is recognized as an important pathogenetic factor in dry eye syndrome, but difficulties in its measurement have limited its utility in the recent past. This prospective, nonrandomized, clinical single-center study investigates the osmolarity in tear samples of patients with keratoconjunctivitis sicca compared with healthy controls.One hundred thirty-three patients [aged 58 years (51–64 years), 86 women and 47 men] with moderate to severe keratoconjunctivitis sicca and 95 controls [aged 52 years (48–61 years), 55 women and 40 men] were enrolled in the trial. Tear samples were collected directly from the inferior lateral tear meniscus. Inclusion criteria were a tear breakup time of less than 5 seconds, a Schirmer test with anesthesia less than 5 mm, and positive symptoms (Ocular Surface Disease Index score > 83). Tear film osmolarity was analyzed by the TearLab osmometer.In our study, patients with moderate to severe keratoconjunctivitis sicca showed a tear film osmolarity of 320 mOsmol/L (301–324 mOsmol/L). The results of the control group were 301 mOsmol/L (298–304 mOsmol/L). Our results revealed a significantly higher tear film osmolarity in patients with moderate to severe keratoconjunctivitis sicca compared with the control group. The sensitivity was 87%, and the specificity was 81%.Our results approved the referent value in moderate to severe dry eye of approximately 316 mOsmol/L, as described in the literature. The results showed a significantly higher tear film osmolarity in patients with severe keratoconjunctivitis sicca compared with the healthy controls. Testing tear film osmolarity can be a very effective objective diagnostic tool in the diagnosis of dry eye disease.