Background: The differentiation of Horner syndrome from physiological anisocoria is important yet clinically challenging. We investigated the diagnostic accuracy of pupillometry to discriminate Horner syndrome from physiological anisocoria compared to pharmacological testing with the alpha-2-agonist apraclonidine, which is considered the current gold standard. Methods: Forty-four adult patients, mostly referred to our neuro-ophthalmology service for evaluation of anisocoria, were included. Automated binocular pupillometry was performed under standardized light conditions before and >30 minutes after instillation of 1% apraclonidine eye drops. A positive apraclonidine test indicating unilateral Horner syndrome was defined as an increase of pupil size in the smaller pupil and decrease of size in the larger pupil. Receiver operator characteristic curves were calculated to find the best pupillometric parameter discriminating Horner syndrome from physiological anisocoria. Results: We found that the parameters measuring the pupillary dilation lag using pupillometry could reliably discriminate Horner syndrome from physiological anisocoria compared to pharmacological testing. Calculating the change of anisocoria at 3–4 seconds after light-off relative to the anisocoria at the end of the light-on period (Δ3-4) may be most suitable to rule out Horner syndrome reaching a sensitivity of 95% and specificity of 68% using a cutoff of 0.35 mm. Conclusions: Our results indicate that pupillometry is a robust tool to measure the dilation lag in Horner syndrome and, therefore, to distinguish pathological from physiological anisocoria obviating pharmacological testing. The high sensitivity of the test will allow to identify the patients with Horner syndrome requiring further investigation.