To increase bioanalytical validity while maintaining sample homogeneity, LC-MS-based profiling of 29 cortical steroids and 6 medullary amines, including catecholamines and metanephrines, in a single run was developed.  A novel LC-MS-based quantitative profiling of steroids, catecholamines, and metanephrines in human serum successfully characterized metabolic features of individual adrenal tumors with Cushing syndrome, primary aldosteronism, and pheochromocytoma/paraganglioma.