Background Cardiac troponins are the preferred biomarkers for the diagnosis of acute myocardial infarction. Although sex‐specific 99th percentile thresholds of troponins are recommended in international guidelines, the clinical effect of their use is poorly investigated. The DANSPOT Study (The Danish Study of Sex‐ and Population‐Specific 99th percentile upper reference limits of Troponin) aims to evaluate the clinical effect of a prospective implementation of population‐ and sex‐specific diagnostic thresholds of troponins into clinical practice. Methods This study is a nationwide, multicenter, stepped‐wedge cluster‐randomized trial of the implementation of population‐ and sex‐specific thresholds of troponins in 22 of 23 clinical centers in Denmark. We established sex‐specific thresholds for 5 different troponin assays based on troponin levels in a healthy Danish reference population. Centers will sequentially cross over from current uniform manufacturer‐derived thresholds to the new population‐ and sex‐specific thresholds. The primary cohort is defined as patients with symptoms suggestive of acute coronary syndrome having at least 1 troponin measurement performed within 24 hours of arrival with a peak troponin value between the current uniform threshold and the new sex‐specific female and male thresholds. The study will compare the occurrence of the primary outcome, defined as a composite of nonfatal myocardial infarction, unplanned revascularization, and all‐cause mortality within 1 year, separately for men and women before and after the implementation of the new sex‐specific thresholds. Conclusions The DANSPOT Study is expected to show the clinical effects on diagnostics, treatment, and clinical outcomes in patients with myocardial infarction of implementing sex‐specific diagnostic thresholds for troponin based on a national Danish reference population. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT05336435.