We prospectively evaluated serum concentrations of glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1), total tau (T-Tau), and neurofilament light (NF-L) from collegiate athletes at baseline and acutely after sport-related concussion (SRC) using the Quanterix Neurology 4Plex "B" (N4PB) multiplex assay. Uninjured controls were matched on age, sex, race, sport, and concussion history. Clinical outcomes included acute symptom severity, balance, rapid automated naming, computerized cognitive testing, and recovery duration. Baseline (n=110; median [IQR] age =19 [18-20] years, 54% male, 61% white/Caucasian) and post-SRC (n=36; median [IQR] age = 19 [18-20] years, 50% male, 61% white/Caucasian) blood samples were analyzed. We observed post-SRC elevations from baseline for GFAP (p=.001, d=1.7), T-Tau (p=.004, d=1.3), and NF-L (p=.010, d=1.1). GFAP (AUC=.958, 95%CI .927-.989, p<.001) and NF-L (AUC=.904, 95%CI .851-.957, p<.001) accurately discriminated SRC from control cases. There were no associations between biomarker concentrations and clinical measurements post-SRC or recovery duration. These findings suggest that, using the multiplex assay, GFAP, T-Tau, and NF-L elevate from baseline acutely after SRC, and both GFAP and NF-L excellently distinguished concussed from control cases. Serum biomarker changes do not necessarily correspond with clinical measurements or recovery duration. Key Words: Concussion, TBI, biomarkers, multiplex, brain injury.