Journal of Medical Internet Research - International Scientific Journal for Medical Research, Information and Communication on the Internet #Preprint #PeerReviewMe: Warning: This is a unreviewed preprint. Readers are warned that the document has not been peer-reviewed by expert/patient reviewers or an academic editor, may contain misleading claims, and is likely to undergo changes before final publication, if accepted, or may have been rejected/withdrawn. Readers with interest and expertise are encouraged to sign up as peer-reviewer, if the paper is within an open peer-review period. Please cite this preprint only for review purposes or for grant applications and CVs (if you are the author). Background: As digital health services are increasingly developing and becoming more interactive in Serbia, a comprehensive instrument for measuring eHealth literacy (EHL) is needed. Objective: This study aimed to translate, culturally adapt, and investigate the psychometric properties of the Serbian version of the eHealth Literacy Questionnaire (eHLQ); to evaluate EHL in the population of primary health care (PHC) users in Serbia; and to explore factors associated with their EHL. Methods: The validation study was conducted in 8 PHC centers in the territory of the Mačva district in Western Serbia. A stratified sampling method was used to obtain a representative sample. The Translation Integrity Procedure was followed to adapt the questionnaire to the Serbian language. The psychometric properties of the Serbian version of the eHLQ were analyzed through the examination of factorial structure, internal consistency, and test-retest reliability. Descriptive statistics were calculated to determine participant characteristics. Differences between groups were tested by the 2-tailed Students t test and ANOVA. Univariable and multivariable linear regression analyses were used to determine factors related to EHL. Results: A total of 475 PHC users were enrolled. The mean age was 51.0 (SD 17.3; range 19-94) years, and most participants were female (328/475, 69.1%). Confirmatory factor analysis validated the 7-factor structure of the questionnaire. Values for incremental fit index (0.96) and comparative fit index (0.95) were above the cutoff of ≥0.95. The root mean square error of approximation value of 0.05 was below the suggested value of ≤0.06. Cronbach α of the entire scale was 0.95, indicating excellent scale reliability, with Cronbach α ranging from 0.81 to 0.90 for domains. The intraclass correlation coefficient ranged from 0.63 to 0.82, indicating moderate to good test-retest reliability. The highest EHL mean scores were obtained for the understanding of health concepts and language (mean 2.86, SD 0.32) and feel safe and in control (mean 2.89, SD 0.33) domains. Statistically significant differences (all P<.05) for all 7 eHLQ scores were observed for age, education, perceived material status, perceived health status, searching for health information on the internet, and occupation (except domain 4). In multivariable regression models, searching for health information on the internet and being aged younger than 65 years were associated with higher values of all domain scores except the domain feel safe and in control for variable age. Conclusions: This study demonstrates that the Serbian version of the eHLQ can be a useful tool in the measurement of EHL and in the planning of digital health interventions at the population and individual level due to its strong psychometric properties in the Serbian context.