Introduction Exogenous Cushing's syndrome is the result of long-term exposure to glucocorticoids, while endogenous Cushing's syndrome occurs due to excessive production of glucocorticoids within the body. Cushing's syndrome remains a diagnostic challenge for the treating physician.Mass spectrometry, with its better resolution, detectability and specificity, paved the way to understanding the cellular and molecular mechanisms involved in the several diseases that facilitated the evolution of biomarkers and personalized medicine, which can be applicable to manage Cushing's syndrome as well.