This study assessed whether S-100β protein could be measured in urine when detectable in plasma after a mild traumatic brain injury (mTBI). Clinical data, plasma and urine samples were collected for the 46 adult patients prospectively enrolled in the emergency department (ED) of a Level 1 trauma center. S-100β protein concentrations were analysed using ELISA. S-100β protein was detectable in 91% and 71% of plasma and urine samples, but values were not correlated (r = 0.002). Urine sampling would have been a non-invasive procedure, but it does not appear to be useful in the ED during the acute phase after an mTBI.