Background: Data correlating dried blood spots (DBS) and plasma concentrations for neonatal biomarkers of brain injury are lacking. We hypothesized that candidate biomarker levels determined from DBS can serve as a reliable surrogate for plasma levels. Methods: In the context of a phase II multi-center trial evaluating erythropoietin for neuroprotection in neonatal encephalopathy (NE), DBS were collected at enrollment (< 24 h), day 2, 4, and 5. Plasma was collected with the first and last DBS. The relationship between paired DBS-plasma determinations of brain-specific proteins and cytokines was assessed by correlation and Bland–Altman analyses. For analytes with consistent DBS-plasma associations, DBS-derived biomarker levels were related to brain injury by MRI and 1-year outcomes. Results: We enrolled 50 newborns with NE. While S100B protein, tumor necrosis factor α, interleukin (IL)1 β, IL-6, IL-8 demonstrated significant DBS-plasma correlations, Bland–Altman plots demonstrated that the methods are not interchangeable, with a 2 to 4-fold error between measurements. No significant relationships were found between DBS levels of TNFα, IL-6, and IL-8 and outcomes. Conclusion: Further work is needed to optimize elution and assay methods before using DBS specimens as a reliable surrogate for plasma levels of candidate brain injury biomarkers in NE.