The routine use of biomarkers, which is also rapidly expanding in the emergency department, carries some potential drawbacks such as the risk of producing false positive results and also places a substantial economical burden on the healthcare system, especially when the use of laboratory resources is poorly discretionary or even inappropriate. The aim of this article is to provide an overview about some general criteria for biomarker validation in the emergency department, and discuss some relevant clinical implications. The leading aspects include analysis of data distribution and diagnostic performance, along with economical and organizational issues. We also brought a pragmatic example, comparing creatine kinase MB, a contemporary-sensitive troponin I and a high-sensitivity troponin I immunoassays for evaluation of patients with suspected acute myocardial infarction at emergency department admission.