Commercially available methylphenidate (MPH) exists as a racemic mixture composed of the d- and l-threo enantiomers. Various pharmacokinetic studies of MPH have shown a greater pharmacological potency of the d-threo enantiomer. Furthermore, it was deduced that the stereoselective cleavage of MPH to produce ritalinic acid (RA) by human carboxylesterase results in a higher oral bioavailability of the d-threo enantiomer. As a requirement for pharmaceutical regulation authorities, efforts have been made to determine the differential biological distribution of d- and l-threo MPH and RA enantiomers. In support of these efforts, numerous analytical procedures have been developed for the chiral separation and quantification of MPH enantiomers in a variety of biological matrices. The available methodologies accomplish the enantioseparation and quantification of MPH using gas chromatography, liquid chromatography or capillary electrophoretic techniques coupled with a variety of detectors. The current review discusses the technical procedures involved, and the sensitivity and selectivity of these assays.