Abnormal elevation of 11beta-HSD1 activities in tissues, such as fat and brain, may contribute to the development of the abdominal obesity and Alzheimer disease, and the inhibition of 11beta-HSD1 might be beneficial to the management of these diseases. To assess the effects of pharmacologic inhibitors of 11beta-HSD1, we developed a fast LC/MS/MS method to quantify corticosteroids in minced tissue samples in the presence of 11beta-HSD substrates. The novel on-line SPE-LC/MS/MS method was developed with dual binary gradient and a throughput of 4.5 min/sample. A total of six corticosteroids (cortisol, cortisone, corticosterone, dehydrocorticosterone, dexamethasone, and dehydrodexamethasone) were studied. The lower limit of quantitation from 0.40 to 11.4 fmol and 4.5 orders magnitude of dynamic range were obtained for these six compounds. Three novel enzymatic bi-products, all isomers of cortisol, were observed in the liver or fat samples. Two of them were identified by matching the HPLC retention times and MS/MS spectra with authentic compounds. The potential interferences of these isomers and their removal are discussed.