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Abstract Background Health-related stigma and its internalization among individuals with chronic health conditions contribute to impaired mental and physical health and quality of life. Research on health-related stigma has been siloed, with disease-specific measures that may not capture the experiences of individuals with multiple health conditions and that prevent comparisons across health conditions. The current study aimed to develop and test a transdiagnostic measure of internalized health-related stigma for use among adults with different physical health conditions. Methods An existing measure of internalized mental health stigma was adapted to assess stigma due to chronic physical health conditions following COSMIN procedures, with input from advisory boards of community members living with a range of stigmatized health conditions (obesity, type 1 and type 2 diabetes, skin diseases, HIV, chronic pain, and cancers) and of health professionals who specialized in these conditions. The new Internalized Health-Related Stigma (I-HEARTS) Scale was tested in an online sample of 300 adults with these health conditions, recruited from ResearchMatch. Additional psychosocial measures of mental health and quality of life were administered, and participants provided information about their health conditions and demographic characteristics. Exploratory factor analysis and tests of reliability and validity were conducted to determine the psychometric properties of the I-HEARTS Scale, and k-means clustering and receiver of characteristic curve analysis were used to determine a clinically meaningful cutoff score indicating high levels of internalized stigma. Results Factor analysis results yielded a 25-item scale with a 3-factor solution, with subscales of Perceived and Anticipated Stigma, Stereotype Application and Self-Devaluation, and Stigma Resistance. Psychometric properties for internal consistency, inter-item and item-total correlations, and test-retest reliability were strong. Certain demographics (e.g., younger age) and characteristics related to health conditions (e.g., greater symptom severity) were associated with higher levels of internalized stigma. I-HEARTS Scale scores correlated moderately to strongly with related but distinct psychosocial measures, and a cutoff score of 3.40 or higher on the 1–7 rating scale was determined to indicate clinically meaningful levels of internalized stigma. Conclusions The I-HEARTS Scale is a reliable and valid measure for the assessment of internalized health-related stigma among adults with varied stigmatized chronic health conditions. Study pre-registration https://osf.io/84c5d/?view_only=87238512f6d6475c87f8f64280a8a15f .