ObjectiveTo compare tear film (TF) osmolarity measured using TearLab® and I‐PEN® osmometers in the same dogs without any ocular surface disease.Animal StudiedFifty‐two dogs (98 eyes) of different breeds were evaluated.ProceduresTear film (TF) osmolarity was evaluated at 2‐min intervals. The test was randomly determined, and single measurements were performed using each osmometer. Subsequently, complete ophthalmologic examinations were performed based on Schirmer tear test‐1 (STT‐1) analysis, tear film breakup time (TFBUT), and slit‐lamp biomicroscopy. For each osmometer, the mean ± standard deviation of the TF osmolarity was calculated, and a paired Student's t‐test was used to compare the values obtained. Pearson correlation analysis was performed to assess the association between osmolarity and other values such as STT‐1, TFBUT, and age.ResultsTear film osmolarity determined using TearLab® (340.42 ± 15.87 mOsm/L) and I‐PEN® (321.58 ± 17.39 mOsm/L) were significantly different (p < .001). However, statistical significance could not be confirmed between osmolarity and other values, such as STT‐1, TFBUT, and age.ConclusionsIn dogs, the TF osmolarity values obtained using TearLab® tend to be higher than those obtained using I‐PEN®, contrary to that observed in humans. These findings can serve as a reference for establishing normal values for each osmometer for clinical use in measuring TF osmolarity in dogs.