Background: With the digitization of health care and the wide availability of Web-based applications, a broad set of skills is essential to properly use such facilities; these skills are called digital health literacy or eHealth literacy. Current instruments to measure digital health literacy focus only on information gathering (Health 1.0 skills) and do not pay attention to interactivity on the Web (Health 2.0). To measure the complete spectrum of Health 1.0 and Health 2.0 skills, including actual competencies, we developed a new instrument. The Digital Health Literacy Instrument (DHLI) measures operational skills, navigation skills, information searching, evaluating reliability, determining relevance, adding self-generated content, and protecting privacy. Objective: Our objective was to study the distributional properties, reliability, content validity, and construct validity of the DHLI’s self-report scale (21 items) and to explore the feasibility of an additional set of performance-based items (7 items). Methods: We used a paper-and-pencil survey among a sample of the general Dutch population, stratified by age, sex, and educational level (T1; N=200). The survey consisted of the DHLI, sociodemographics, Internet use, health status, health literacy and the eHealth Literacy Scale (eHEALS). After 2 weeks, we asked participants to complete the DHLI again (T2; n=67). Cronbach alpha and intraclass correlation analysis between T1 and T2 were used to investigate reliability. Principal component analysis was performed to determine content validity. Correlation analyses were used to determine the construct validity. Results: Respondents (107 female and 93 male) ranged in age from 18 to 84 years (mean 46.4, SD 19.0); 23.0% (46/200) had a lower educational level. Internal consistencies of the total scale (alpha=.87) and the subscales (alpha range .70-.89) were satisfactory, except for protecting privacy (alpha=.57). Distributional properties showed an approximately normal distribution. Test-retest analysis was satisfactory overall (total scale intraclass correlation coefficient=.77; subscale intraclass correlation coefficient range .49-.81). The performance-based items did not together form a single construct (alpha=.47) and should be interpreted individually. Results showed that more complex skills were reflected in a lower number of correct responses. Principal component analysis confirmed the theoretical structure of the self-report scale (76% explained variance). Correlations were as expected, showing significant relations with age (ρ=–.41, P<.001), education (ρ=.14, P=.047), Internet use (ρ=.39, P<.001), health-related Internet use (ρ=.27, P<.001), health status (ρ range .17-.27, P<.001), health literacy (ρ=.31, P<.001), and the eHEALS (ρ=.51, P<.001). Conclusions: This instrument can be accepted as a new self-report measure to assess digital health literacy, using multiple subscales. Its performance-based items provide an indication of actual skills but should be studied and adapted further. Future research should examine the acceptability of this instrument in other languages and among different populations.