A high-sensitivity cardiac troponin T (hs-cTnT) assay was developed and validated to improve the detection of cardiac troponin T in the diagnosis of myocardial infarction (MI). This assay is a modification of the Roche fourth-generation cTnT assay, with improvements in sensitivity and reduced susceptibility to interferences. The analytical range of the hs-cTnT assay is 3 to 10,000 ng/L, with a 99th percentile cutoff of 13.5 ng/L. The assay demonstrated a coefficient of variation (CV) of 9% at this cutoff, indicating good precision. The assay showed high specificity for cTnT, with no significant cross-reactivity with other cardiac or skeletal muscle proteins. It was also tested for interference with various substances, including hemoglobin, and showed no significant interference up to 1000 mg/L hemoglobin. The hs-cTnT assay was compared with the fourth-generation cTnT assay in clinical settings. It identified more patients with non-ST elevation myocardial infarction (non-STEMI) on presentation and had a shorter time to diagnosis. The hs-cTnT assay showed better performance in detecting low concentrations of cTnT, which are associated with cardiovascular risk factors and myocardial dysfunction. The assay was standardized against the fourth-generation cTnT assay and demonstrated excellent correlation across the entire measuring range. The hs-cTnT assay complies with the ESC-ACCF-AHA-WHF recommendations for the use of cardiac troponin in the diagnosis of MI. It provides improved sensitivity and precision, allowing for earlier detection of MI and better differentiation between ischemic and non-ischemic causes of cTn elevation. However, the use of a lower decision cutpoint may reduce diagnostic specificity, emphasizing the need for adherence to the universal MI definition. Future studies are needed to evaluate the clinical significance of low concentration elevations of hs-cTnT in patients with acute coronary syndrome.A high-sensitivity cardiac troponin T (hs-cTnT) assay was developed and validated to improve the detection of cardiac troponin T in the diagnosis of myocardial infarction (MI). This assay is a modification of the Roche fourth-generation cTnT assay, with improvements in sensitivity and reduced susceptibility to interferences. The analytical range of the hs-cTnT assay is 3 to 10,000 ng/L, with a 99th percentile cutoff of 13.5 ng/L. The assay demonstrated a coefficient of variation (CV) of 9% at this cutoff, indicating good precision. The assay showed high specificity for cTnT, with no significant cross-reactivity with other cardiac or skeletal muscle proteins. It was also tested for interference with various substances, including hemoglobin, and showed no significant interference up to 1000 mg/L hemoglobin. The hs-cTnT assay was compared with the fourth-generation cTnT assay in clinical settings. It identified more patients with non-ST elevation myocardial infarction (non-STEMI) on presentation and had a shorter time to diagnosis. The hs-cTnT assay showed better performance in detecting low concentrations of cTnT, which are associated with cardiovascular risk factors and myocardial dysfunction. The assay was standardized against the fourth-generation cTnT assay and demonstrated excellent correlation across the entire measuring range. The hs-cTnT assay complies with the ESC-ACCF-AHA-WHF recommendations for the use of cardiac troponin in the diagnosis of MI. It provides improved sensitivity and precision, allowing for earlier detection of MI and better differentiation between ischemic and non-ischemic causes of cTn elevation. However, the use of a lower decision cutpoint may reduce diagnostic specificity, emphasizing the need for adherence to the universal MI definition. Future studies are needed to evaluate the clinical significance of low concentration elevations of hs-cTnT in patients with acute coronary syndrome.