Objectives The study objective was to validate a new high-sensitivity troponin I (hs-TnI) assay in a clinical protocol for assessing patients who present to the emergency department with chest pain. Background Protocols using sensitive troponin assays can accelerate the rule out of acute myocardial infarction in patients with low-risk (suspected) acute coronary syndrome (ACS). Methods This study evaluated 2 prospective cohorts of patients in the emergency department with ACS in an accelerated diagnostic pathway integrating 0- and 2-h hs-TnI results, Thrombolysis In Myocardial Infarction (TIMI) risk scores, and electrocardiography. Strategies to identify low-risk patients incorporated TIMI risk scores = 0 or =1. The primary endpoint was a major adverse cardiac event (MACE) within 30 days. Results In the primary cohort, 1,635 patients were recruited and had 30-day follow-up. A total of 247 patients (15.1 ) had a MACE. The finding of no ischemic electrocardiogram and hs-TnI =26.2 ng/l with the TIMI = 0 and TIMI =1 pathways, respectively, classified 19.6 (n = 320) and 41.5 (n = 678) of these patients as low risk; 0 (n = 0) and 0.8 (n = 2) had a MACE, respectively. In the secondary cohort, 909 patients were recruited. A total of 156 patients (17.2 ) had a MACE. The TIMI = 0 and TIMI =1 pathways classified 25.3 (n = 230) and 38.6 (n = 351), respectively, of these patients as low risk; 0 (n = 0) and 0.8 (n = 1) had a MACE, respectively. Sensitivity, specificity, and negative predictive value for TIMI = 0 in the primary cohort were 100 (95 confidence interval [CI]: 98.5 to 100 ), 23.1 (95 CI: 20.9 to 25.3 ), and 100 (95 CI: 98.8 to 100 ), respectively. Sensitivity, specificity, and negative predictive value for TIMI =1 in the primary cohort were 99.2 (95 CI: 97.1 to 99.8), 48.7 (95 CI: 46.1 to 51.3), and 99.7 (95 CI: 98.9 to 99.9), respectively. Sensitivity, specificity, and negative value for TIMI =1 in the secondary cohort were 99.4 (95 CI: 96.5 to 100), 46.5 (95 CI: 42.9 to 50.1), and 99.7 (95 CI: 98.4 to 100), respectively. Conclusions An early-discharge strategy using an hs-TnI assay and TIMI score =1 had similar safety as previously reported, with the potential to decrease the observation periods and admissions for approximately 40 of patients with suspected ACS. (Advantageous Predictors of Acute Coronary Syndromes Evaluation [APACE] Study, NCT00470587; A 2hr Accelerated Diagnostic Protocol to Assess patients with chest Pain symptoms using contemporary Troponins as the only biomarker [ADAPT]: A prospective observational validation study, ACTRN12611001069943) ? 2013 by the American College of Cardiology Foundation Published by Elsevier Inc.