It is postulated that elevated tissue concentrations of cortisol may be associated with the development of metabolic syndrome, obesity, and type 2 diabetes. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme regenerates cortisol from inactive cortisone in tissues such as liver and adipose. To better understand the pivotal role of 11β-HSD1 in disease development, an in vivo microdialysis assay coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using stable isotope-labeled (SIL) cortisone as a substrate was developed. This assay overcomes the limitations of existing methodologies that suffer from radioactivity exposure and analytical assay sensitivity and specificity concerns. Analyte extraction efficiencies (E d ) were evaluated by retrodialysis. The conversion of SIL-cortisone to SIL-cortisol in rhesus monkey adipose tissue was studied. Solutions containing 100, 500, and 1000ng/mL SIL-cortisone were locally delivered through an implanted 30-mm microdialysis probe in adipose tissue. At the delivery rate of 1.0 and 0.5μL/min, E d values for SIL-cortisone were between 58.7±5.6% (n=4) and 72.7±1.3% (n=4), whereas at 0.3μL/min E d reached nearly 100%. The presence of 11β-HSD1 activities in adipose tissue was demonstrated by production of SIL-cortisol during SIL-cortisone infusion. This methodology could be applied to cortisol metabolism studies in tissues of other mammalian species.