This review article underlines the importance of stable isotope dilution mass spectrometry analysis of endogenous and synthetic corticosteroids for the pharmacokinetic and in vivo metabolic investigations in humans. The multi-labeled corticosteroids with stable isotopes such as [1,2,4,19-13C4]cortisol, [1,2,3,4-13C4]cortisol, [1,1,19,19,19- 2H5]cortisol, [9,11,12,12-2H4]cortisol, etc. have been used not only as analytical internal standard for the mass spectrometry but as tracer in the pharmacokinetic and metabolic studies in human in vivo. Stable isotope dilution analysis of cortisol metabolites (tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), tetrahydrocortisone (THE), and 6β - hydroxycortisol (6β-OHF)) and synthetic corticosteroids (prednisolone, prednisone, budesonide, fluticasone, etc.) is also the subject of this review. In the gas chromatographic mass spectrometric (GC-MS) analysis of corticosteroids, it is usual to employ derivatives in which some or all of the original functional groups are protected. Thermal stabilization of corticosteroids in the analysis has been achieved most successfully by formation of methoxime-trimethylsilyl (MO-TMS) derivatives. Bismethylenedioxy-pentafluoropropionyl (BMD-PFP) derivatives have been developed to simultaneously measure cortisol, cortisone, and their tetrahydrocorticoid metabolites (THF, allo-THF, and THE) and/or prednisolone and prednisone in plasma and urine. Stable isotope tracer methodology has been applied to the measurements of cortisol production rate, determination of human urinary cortisol metabolites, and assessment of in vivo activities of 11β- hydroxysteroid dehydrogenase (11β-HSD) in humans. This methodology also has been used for the validity of endogenous cortisol 6β-hydroxylation clearance as a new index for phenotyping the in vivo cytochrome P450 3A (CYP3A) in humans.