Background Cortisol plays a role in the regulation of metabolic homeostasis. Dexamethasone is a powerful synthetic glucocorticoid that acts as an anti-inflammatory and immunosuppressive agent. Plasma free cortisol, which has a low concentration in the blood, is the cortisol fraction that provides biological activity. It is emphasized that measurement of dexamethasone, total and free cortisol levels can directly affect the results of dexamethasone suppression test, which is routinely used to exclude endogenous hypercortisolemia in patients with adrenal adenoma, and is important in differential diagnosis. Purpose In this study, a precise and rapid method was developed for the measurement of dexamethasone, total and free cortisol levels in plasma samples, besides demonstrating the clinical applicability of the method with samples from adrenal adenoma patients. Methods Method optimization studies were performed using liquid chromatography-tandem mass spectrometry with stable isotope dilution-multiple reaction monitoring. Results The assay demonstrated a good linear dynamic range of 0.5–20 μg/L, 5–200 μg/L and 0.5–100 μg/L for dexamethasone, total and free cortisol, respectively. The values for intra- and inter-day precisions of analytes were ≤6.9% with the accuracies ranging from 91.6% to 113.0%. Conclusion Measurement of these parameters can be used reliably to diagnose diseases causing hypercortisolemia.