Glial fibrillary acidic protein (GFAP) and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) may aid in the evaluation of traumatic brain injury (TBI). The objective of this analysis was to compare GFAP and UCH-L1 values measured using a handheld device compared with a core laboratory platform. We analyzed plasma samples from patients with TBI and healthy controls enrolled in the Transforming Research and Clinical Knowledge in TBI (TRACK-TBI) cohort study. GFAP and UCH-L1 were measured twice in each subject using prototype assays, first with the Abbott i-STAT™ handheld device, and second with the Abbott ARCHITECT^® platform. We then quantified the agreement in biomarker values obtained using these two methods. GFAP and UCH-L1 were measured twice in 570 and 572 samples, respectively. GFAP values measured by the ARCHITECT platform (median 143.3 [interquartile range (IQR): 19.8-925.8] pg/mL) were higher than values measured by the i-STAT (median 116.0 [IQR: 9.2-856.5] pg/mL). GFAP values from the two platforms were strongly correlated (p = 0.985). Similarly, UCH-L1 values measured by the ARCHITECT platform (median 163.9 [IQR: 82.5-412.4] pg/mL) were higher than values measured by the i-STAT (median 122.5 [IQR: 63.0-297.3] pg/mL). UCH-L1 values from the two platforms were strongly correlated (p = 0.933). Passing-Bablok regression equations were developed to estimate the relationship between the two platforms, specifically to predict i-STAT values from the ARCHITECT platform. GFAP and UCH-L1 values measured using the prototype assays on the Abbott i-STAT and ARCHITECT platforms are strongly correlated and values from either platform may be converted to the other.