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To assess the effect of examiner handedness on intraocular pressure (IOP) readings using the Tono-Pen XL in eyes without corneal pathology or previous ocular surgeries.Patients 18 years of age or older were included in this prospective study. Participants who had a history of corneal conditions or other characteristics that would prevent reliable IOP measurements were excluded. Five experienced examiners, 2 right-hand dominant and 3 left-hand dominant, took bilateral IOP measurements with a Tono-Pen XL. Each participant was tested by 2 examiners, 1 left-handed and 1 right-handed, in a random order. The time between each examiner’s measurements was 15 minutes. A paired t test was used to compare IOP differences between “near” (right eye for right-hand or left eye for left-hand dominant examiner) and “far” (right eye by left-hand or left eye by right-hand dominant examiner) measurements for each eye.Forty-six participants with a mean age of 33.8 years (±12.8) were enrolled. No significant difference in IOP between near and far eyes was found (IOP difference=IOPfar–IOPnear=−0.11 [±2.74] mm Hg; P=0.70). Right-hand dominant examiners consistently measured significantly higher IOP (0.83±2.03 mm Hg; P=0.008) compared with left-hand dominant examiners, regardless of the relative position of the eye with respect to the examiner.The results suggest that IOP readings are not influenced by the positioning of the patient relative to the dominant hand of the examiner.