Introduction: Ruling out acute myocardial infarction (AMI) using serial troponin testing is central to the care of many emergency department (ED) patients with chest pain. While diagnostic strategies using conventional troponin assays require repeat sampling over many hours to avoid missed diagnoses, serial high-sensitivity troponin (hs-cTn) assays may be able to exclude AMI in most patients within 1 or 2 hours. However, many of the initial studies deriving and validating these rapid diagnostic algorithms had all hs-cTn samples analyzed in a central core lab likely representing optimal assay performance. This objective of this study is to validate a 2-hour rapid diagnostic algorithm to exclude AMI in ED chest pain patients using an hs-cTn assay in real world practice. Methods: This prospective cohort study was conducted at a single urban tertiary center and regional percutaneous coronary intervention site in Calgary, Alberta. Patients were eligible for enrolment if they presented to the ED with chest pain, were 25-years or older and required biomarker testing to rule out AMI at the discretion of the attending emergency physician. Patients were excluded if they had clear acute ischemic ECG changes, new arrhythmia or renal failure requiring hemodialysis. A high-sensitivity troponin result (Roche Elecsys hs-cTnT) was obtained in all patients at ED presentation and 2-hours later. The primary outcome was AMI on the index visit. Secondary outcomes included 30-day AMI and 30-day major adverse cardiac events (MACE - including AMI, revascularization or cardiac death). Electronic medical records were reviewed and telephone follow-up was obtained for all patients at 30-days to ensure relevant events were captured. Two physician adjudication (board-certified emergency physician and cardiologist) was obtained for all outcomes. This study was REB approved. Results: A total of 549 patients were enrolled from August 2014 September 2016 with 2-hour serial hs-cTnT results, of which 349 (63.6%) met the 2-hour rapid diagnostic algorithm low risk criteria (time 0 h/2 h hs-cTnT <14 ng/L and delta 2 h <4 ng/L). The sensitivity of the 2-hour low risk criteria for index AMI was 98.4% (95% CI 91.3%-100%) and for 30-day AMI was 98.4% (95% CI 91.6-100%). The sensitivity for 30day MACE was lower 84.4% (95% CI 74.4-91.7%) but maintained a high negative predictive value, 96.6% (95% CI 94.1-98.2%). Conclusion: A 2-hour rapid diagnostic algorithm using an hs-cTnT assay was highly sensitive for AMI on the index visit and successfully identified patients at low risk of 30-day AMI. Sensitivity for MACE was lower, reminding us that while biomarker-only rapid diagnostic algorithms excel at ruling out AMI, careful clinical risk stratification is needed to avoid missed MACE events.