Tramadol was found in a man's hair sample during an abstinence test necessary to regain his driving license. The suspect denied having taken tramadol claiming external contamination as the reason for the positive result, as he was working in a tramadol production company. Nevertheless, low concentrations of both major metabolites, N-desmethyltramadol (NDMT) and O-desmethyltramadol (ODMT), were found in hair (180 and 6 pg/mg hair, respectively). To assess this case, tramadol concentrations and metabolite to parent drug concentration ratios were determined in hair samples of 75 patients taking tramadol and of eight employees working in the production and laboratory site of the same company. Additionally, wash water used for decontaminating hair was analyzed for both groups, patients and employees. Analysis of hair sample extracts was performed by LC-MS/MS using multiple reaction monitoring (MRM), information dependent acquisition (IDA) and enhanced product ion scan (EPI). High variations of metabolite to parent drug concentration ratios in hair samples of patients were observed. Differences in NDMT and ODMT to tramadol concentration ratios were found when comparing the cohort of patients to employees. The suspect could be included in the cohort of employees considering the ODMT to tramadol concentration ratio in hair and tramadol concentration ratio in wash water versus hair. Metabolite to parent drug concentration ratios of hair samples may represent a helpful tool for the differentiation of tramadol intake versus external contamination. Ratios of tramadol concentrations in wash water versus the subjects' hair may provide additional information for case assessments.