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.