This study reports the development and validation of a novel high-sensitivity cardiac troponin T (hs-cTnT) assay, a modification of the Roche fourth-generation cTnT assay. The validation included testing analytical sensitivity, specificity, interferences, and precision. The assay had an analytical range from 3 to 10,000 ng/L, with a 99th percentile cutoff of 13.5 ng/L and a coefficient of variation (CV) of 9%. The assay was highly specific for cTnT, showing no interference from human cTnI, cTnC, skeletal muscle TnT, or hemoglobin concentrations up to 1000 mg/L. Clinically, the hs-cTnT assay identified a significantly higher number of patients with non-ST elevation myocardial infarction (non-STEMI) on presentation compared to the fourth-generation cTnT assay (45 vs 20 patients, P = 0.0004). The time to diagnosis was also significantly shorter with the hs-cTnT assay (mean 71.5 min vs 246.9 min, P < 0.01). The analytical performance of the hs-cTnT assay complies with the ESC-ACCF-AHA-WHF Global Task Force recommendations for use in the diagnosis of myocardial infarction (MI).This study reports the development and validation of a novel high-sensitivity cardiac troponin T (hs-cTnT) assay, a modification of the Roche fourth-generation cTnT assay. The validation included testing analytical sensitivity, specificity, interferences, and precision. The assay had an analytical range from 3 to 10,000 ng/L, with a 99th percentile cutoff of 13.5 ng/L and a coefficient of variation (CV) of 9%. The assay was highly specific for cTnT, showing no interference from human cTnI, cTnC, skeletal muscle TnT, or hemoglobin concentrations up to 1000 mg/L. Clinically, the hs-cTnT assay identified a significantly higher number of patients with non-ST elevation myocardial infarction (non-STEMI) on presentation compared to the fourth-generation cTnT assay (45 vs 20 patients, P = 0.0004). The time to diagnosis was also significantly shorter with the hs-cTnT assay (mean 71.5 min vs 246.9 min, P < 0.01). The analytical performance of the hs-cTnT assay complies with the ESC-ACCF-AHA-WHF Global Task Force recommendations for use in the diagnosis of myocardial infarction (MI).