The steps involved in the methods for the determination of vitamin D 3 metabolites (namely, 25-hydroxyvitamin D 3 , 1,25-dihydroxyvitamin D 3 , 24,25-dihydroxyvitamin D 3 ) mainly in clinical samples are critically reviewed. Sample pretreatment (e.g. deproteinization, saponification, liquid–liquid and liquid–solid extraction, etc.) as a function of both type of sample and detection system, quantitation based on protein saturation and liquid as well as gas chromatography are discussed. The chemical principles on which the methods are based and the derivatization procedures, which facilitate separation and/or detection, are also commented upon. Finally, the future prospects of the research on methods for the determination of these metabolites are outlined.