Adrenal steorids and catecholamines are produced by cortex and medullar of human adrenal glands, respectively.  These hormones are mainly associated with physiology and disease progression of adrenal incidentalomas, including Cushing’s syndrome, primary aldosteroism and pheochromocytoma, which are required several steps for discriminating individual diseases.  Here, LC-MS based simultaneous profiling has been developed to measure 29 adrenal steroids and 6 catecholamines for the differential diagnosis of adrenal incidentalomas in one-step monitoring.  For the simultaneous profiling of hydrophilic catecholamines and liphophylic adrenal steroids, catecholamines were derivatized using isobutyl chloroformate.  The calibration linearities were 0.9565 ~ 0.9982 (0.01 – 500 ng/mL for adrenal steroids) and 0.9197 ~ 0.9958 (0.1 – 25 ng/mL for catecholamines).  The LOQs ranged from 0.01 to 0.1 ng/mL and from 0.1 to 0.5 ng/mL for adrenal steroids and catecholamines, respectively.  Accuracy (% bias) and precision (% CV) were 93.5% ~ 112.0% and 3.5% ~ 12.9%, respectively, for all adrenal hormones studied.  A further study on additional method validation is in progress to bring this method into the identification of almost all clinically relevant adrenal tumors.