Traumatic brain injury (TBI) is a major epidemic known as the aftermath of a large force exerted on the head. In the U.S., the vast majority of TBI cases are classified as mild TBI (mTBI). A primary method to diagnose mTBI is computerized tomography (CT) scans; however, the scans lack the sensitivity to detect more subtle forms of brain injury. Thus, there is an apparent need for a quick, biofluid-based diagnostic test for mTBI. The present study develops an ultra-sensitive assay to test for ubiquitin C-terminal hydrolase-L1 (UCH-L1), a protein mainly found in neuronal cell body cytoplasm, as a biomarker. Previous literature suggests that UCH-L1 is released from injured neurons and then enters cerebrospinal fluid (CSF) following CNS injuries and neurodegeneration. The methodology entailed screening various antibodies to determine a successful capture and detector pair, optimizing the conditions (i.e., diluent type and antibody concentrations), and then testing the CSF, serum, and plasma samples from rat, mouse, and human subjects. We conducted the analyses in the order of Western Blot, regular sandwich ELISA protocol, MSD high-sensitive homebrew ELISA development, and Quanterix ultra-sensitive homebrew ELISA development. The study proved that participants with TBI showed increased levels of UCH-L1 in their CSF.