Biomarkers are key tools and can provide crucial information on the complex cascade of events and molecular mechanisms underlying traumatic brain injury (TBI) pathophysiology. Obtaining a profile of distinct classes of biomarkers reflecting core pathologic mechanisms could enable us to identify and characterize the initial injury and the secondary pathologic cascades. Thus, they represent a logical adjunct to improve diagnosis, track progression and activity, guide molecularly targeted therapy, and monitor therapeutic response in TBI. Accordingly, great effort has been put into the identification of novel biomarkers in the past 25 years. However, the role of brain injury markers in clinical practice has been long debated, due to inconsistent regulatory standards and lack of reliable evidence of analytical validity and clinical utility. We present a comprehensive overview of the markers currently available while characterizing their potential role and applications in diagnosis, monitoring, drug discovery, and clinical trials in TBI. In reviewing these concepts, we discuss the recent inclusion of brain damage biomarkers in the diagnostic guidelines and provide perspectives on the validation of such markers for their use in the clinic.