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Despite improvements in the process of organ donation and transplants, the number of organ donors is progressively declining in developed countries. Therefore, the early detection of patients at risk for brain death (BD) is a priority for transplant teams seeking more efficient identification of potential donors. In the extensive literature on S100B as a biomarker for traumatic brain injury (TBI), no evidence appears to exist on its prognostic capacity as a predictor of BD after severe TBI. The objective of this study is to assess the value of including acute S100B levels in standard clinical data as an early screening tool for BD after severe TBI. This prospective study included patients with severe TBI (Glasgow Coma Scale score [GCS] £ 8) admitted to our Neurocritical Care Unit over a 30 month period. We collected the following clinical variables: age, gender, GCS score, pupillary alterations at admission, hypotension and pre-hospital desaturation, CT scan results, isolated TBI or other related injuries, Injury Severity Score (ISS), serum S100B levels at admission and 24 h post-admission, and a final diagnosis regarding BD. Of the 140 patients studied, 11.4% developed BD and showed significantly higher S100B concentrations ( p < 0.001). Multivariate analysis showed that bilateral unresponsive mydriasis at admission and serum S100B at 24 h post-admission had odds ratios (ORs) of 21.35 ( p = 0.005) and 4.9 ( p = 0.010), respectively. The same analysis on patients with photomotor reflex in one pupil at admission left only the 24 h S100B sample in the model (OR = 15.5; p = 0.009). Receiver operating characteristics (ROC) curve analysis on this group showed the highest area under the curve (AUC) (0.86; p = 0.001) for 24 h S100B determinations. The cut off was set at 0.372 lg/L (85.7% sensitivity, 79.3% specificity, positive predictive value [PPV] = 18.7% and negative predictive value [NPV] = 98.9%). This study shows that pupillary responsiveness at admission, as well as 24 h serum S100B levels, could serve as screening tools for the early detection of patients at risk for BD after severe TBI.