An individual’s nervous and cognitive systems are lateralized, and handedness represents a behavioral mani- festation of such organization. Therefore, accurately and reliably measuring handedness has repercussion on our understanding of both the human brain and cognition. The Edinburgh Handedness Inventory (EHI) is the most frequently used instrument to measure handedness both in clinical practice and research. We assessed the psychometric properties of the Spanish version of the EHI in a sample of 348 Chilean university students by confirmatory factor analysis. Cronbach’s alpha and composite reliability were calculated to evaluate the internal consistency and reliability of the EHI, while the average variance extracted was estimated to evaluate its con- vergent validity. A 10-item unifactorial structure was confirmed, with factor loadings ≥0.50, showing excellent goodness-of-fit indicators, very high internal consistency and adequate composite reliability and convergent validity. Socio-demographic variables (sex, area of residence and belonging to an indigenous people or com- munity) did not significantly modulate the EHI scores. Overall, by using this validated version of the EHI to accurately and reliably measure handedness in the greater Spanish population, researchers will be able to produce robust data to tackle the still open questions of lateralization in human cognitive and neural archi- tecture.