Biomarkers complement clinical assessment and the 12-lead ECG in the diagnosis, risk stratification, triage, and management of patients with suspected acute coronary syndrome (ACS). While there is broad consensus that cardiac troponin (cTn) I or T is the preferred biomarker in clinical practice, important uncertainties remain regarding the value of high-sensitivity cTn assays, their best clinical use including the most appropriate timing of serial measurements, as well as the added value of other biomarkers reflecting and quantifying other pathophysiological signals including copeptin and natriuretic peptides. This review will address these aspects with a focus on the diagnostic application of biomarkers, as they are associated with immediate therapeutic consequences. In addition, this review will briefly highlight that increased diagnostic accuracy offered by high-sensitivity cTn assays has contributed to improve our understanding of the incidence, pathophysiology, and mortality of the two distinct components currently summarized under the term ACS: acute myocardial infarction and unstable angina.