The objective of the study was to examine how S100B, a biomarker of traumatic brain injury (TBI), contributes to outcome prediction after adjusting for known parameters, including age, Glasgow Coma Scale (GCS), pupil reaction, and computed tomography (CT) variables; to examine which parameters have the best correlation to elevated serum levels of S100B; and to investigate when to sample S100B to achieve the strongest association to outcome. This retrospective study included 265 patients with TBI admitted to the neurointensive care unit, Karolinska University Hospital Solna, Stockholm, Sweden. Univariate and multivariate proportional odds regressions were performed to determine parameters most closely related to outcome, and how S100B adds to prediction accuracy. Age (p<0.0001), pupil reaction (p<0.0001), and levels of S100B (p<0.0001) had the strongest statistical correlation to outcome. The area under curve of S100B, the first 48 h after trauma, yielded an additional explained variance of 6.6% in excess of known outcome parameters, including age, GCS, pupil reaction, and CT variables, themselves exhibiting an explained variance of 29.3%. S100B adds substantial information regarding patient outcome, in excess of that provided by known parameters. Only CT variables were found to be significant predictors of increased levels of S100B in uni- and multivariate analysis. Early samples of S100B, within 12 h after trauma, appear to have little prognostic value, and S100B should likely be sampled 12-36 h following trauma to best enhance TBI outcome prediction.