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A chiral LC–MS/MS assay for ketamine and norketamine was developed and validated.Analytical run time was just 5 min per injection.High sensitivity allowed sequential blood collection from an individual mouse.This method successfully revealed stereoselective profiles in mouse plasma and CNS.This method would be useful for studying antidepressant actions of ketamine.A novel method for the rapid and sensitive chiral determination of ketamine and norketamine in mouse plasma, brain and cerebrospinal fluid (CSF) was developed using liquid chromatography-tandem mass spectrometry (LC–MS/MS). This method reduces the required matrix volume, compared with a previously reported chiral assay method for ketamine and norketamine. The method involves the deproteinization of a small amount of biological matrix (corresponding to 5 μL of plasma, 10 mg of brain, or 2.5 μL of CSF) using a water-miscible organic solvent containing 2H4-norketamine as an internal standard, the direct injection of the organic supernatant into an LC–MS/MS system, chiral separation on a CHIRALPAK AS-3R column (4.6 mm i.d. × 100 mm, 3 μm particles), and detection by electrospray ionization-selected reaction monitoring with an analytical run time of 5 min. The lower limits of quantification for ketamine and norketamine enantiomers were 1 ng/mL (plasma), 0.5 ng/g (brain) and 2 ng/mL (CSF). A good linearity of the calibration curves was obtained within a range of 1000-fold. The newly developed method was successfully used to determine the concentrations of ketamine and norketamine in mouse samples (plasma, brain and CSF) in a stereoselective manner. Therefore, this method is expected to contribute to the elucidation of the roles of ketamine and its metabolites in the antidepressant actions of ketamine.