Ketamine (KT), primarily used as a general anaesthetic agent in clinical practice, has become very popular in recent years as a recreational drug, due to its dissociative and hallucinogenic effects. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method has been developed and validated for the quantification of KT and its main metabolite norketamine (NK) in 2.0mg of hair. Sample preparation consisted of a rapid, simultaneous pulverization and extraction step in acidic solution, followed by centrifugation and filtration. Gradient elution was performed by an Atlantis T3 analytical column, and deuterated KT was used as the internal standard. Positive ion electrospray ionization and HRMS determination in full-scan mode were achieved with an Orbitrap mass spectrometer. The method has a linear range of 0.05-50 ng/mg, a limit of quantisation of 0.05 ng/mg and a limit of detection of 0.02 ng/mg for both KT and NK. The validated method was applied for the determination of KT and NK in two authentic hair samples from subjects suspected of taking psychoactive substances. The detection of the metabolite at low concentration gave proof for systemic drug origin and an investigation into the possible presence of further metabolites was performed by means of retrospective screening.