Introduction <p>Adopting a learning health system (LHS) approach holds promise for bridging knowledge between policymakers, health professionals, managers, researchers, and patients and their families to collaboratively improve health care. Organizational readiness assessments exist in the quality improvement literature, but may not consider LHS components. This study aimed to establish the content validity of a new LHS readiness questionnaire.</p> Methods <p>A three-round Delphi study was conducted to establish consensus on the importance, relevance, clarity, and comprehensiveness of the domains and items included in this questionnaire. Purposive sampling was used to recruit participants with expertise in LHS who are involved in healthcare organizations across Canada and internationally (<i>n</i> = 41). A minimum of 70% agreement represented consensus. A steering committee reviewed findings and refined items for clarity. Modified items were re-tested in subsequent rounds.</p> Results <p>In Round 1, 85 items were tested, of which 41 achieved consensus, 7 were removed, 21 underwent major modification, 16 were clarified and retested, and 11 new items were proposed. Round 2 tested 36 items (25 revised, 11 new). 18 items achieved consensus, 8 were removed, and 10 were modified. In Round 3, 10 items were tested, 5 achieved consensus, 1 was removed, and 4 were clarified and included post-expert panel discussion. Overall, 41 items were retained in their original form, 20 were modified, and 7 new items were added. The final measure includes 68 items reflected by four domains: (1) performance to data (<i>n</i> = 13 items), (2) data to knowledge (<i>n</i> = 13 items), (3) knowledge to performance (<i>n</i> = 22 items), and (4) LHS core values (<i>n</i> = 20 items).</p> Conclusion <p>The proposed new measure can help establish organizational readiness for change. Future research should seek to test the psychometric properties of this tool and explore potential barriers to its adoption amongst interested parties.</p>