Past and recent attempts at devising objective biomarkers for traumatic brain injury (TBI) in both blood and cerebrospinal fluid have focused on abundance measures of time-dependent proteins. Similar independent determinants would be most welcome in diagnosing the most common form of TBI, mild TBI (mTBI), which remains difficult to define and confirm based solely on clinical criteria. There are currently no consensus diagnostic measures that objectively define individuals as having sustained an acute mTBI. Plasma metabolomic analyses have recently evolved to offer an alternative to proteomic analyses, offering an orthogonal diagnostic measure to what is currently available. The purpose of this study was to determine whether a developed set of metabolomic biomarkers is able to objectively classify college athletes sustaining mTBI from non-injured teammates, within 6 hours of trauma and whether such a biomarker panel could be effectively applied to an independent cohort of TBI and control subjects. A 6-metabolite panel was developed from biomarkers that had their identities confirmed using tandem mass spectrometry (MS/MS) in our Athlete cohort. These biomarkers were defined at ≤6 hours following mTBI and objectively classified mTBI athletes from teammate controls, and provided similar classification of these groups at the 2, 3, and 7 days post-mTBI. The same 6-metabolite panel, when applied to a separate, independent cohort provided statistically similar results despite major differences between the two cohorts. Our confirmed plasma biomarker panel objectively classifies acute mTBI cases from controls within 6 hours of injury in our two independent cohorts. While encouraged by our initial results, we expect future studies to expand on these initial observations.