The use of the drug scopolamine in drug-facilitated crimes is known. Nevertheless, given the high potency of the drug and its rapid metabolism, analysis in blood and urine may not be sufficient for drug detection in late crime declaration, especially following a single-dose administration in drug-facilitated sexual assault (DFSA) cases. Hair may constitute an essential supplemental matrix extending the drug detection window in such cases. This case report presents quantitative data on scopolamine findings in urine and hair in a DFSA case. A young female had consumed several alcoholic drinks at a party venue when her behaviour became noticeably peculiar. Later, she woke up next to an unknown man and had no recollection of the night's events. Blood and urine samples were collected 18 h after the incident. The initial toxicological target screening using UHPLC–TOF-MS detected scopolamine in the hydrolysed urine sample, and quantification yielded 41 µg/L scopolamine in urine, while blood was negative. Segmental hair analysis using multitarget UHPLC–MS/MS was performed on three washed 2-cm segments of hair collected five weeks after the incident, yielding 0.37 pg/mg scopolamine only in the relevant hair segment. This case report provides novel insight into the concentration in hair following a single exposure of scopolamine and the feasibility of detecting scopolamine in hair by comparison to published toxicological findings.