Synthetic glucocorticoids (GCs) are widely used in clinical therapeutic and diagnostic purpose, but serious adverse effects caused by inappropriate and prolonged GC treatment are considerable in clinics.  In particular, dexamethasone suppression test (DST), which is the first-tier test for excessive cortisol production, may be complicated by a combination of comorbidities.  The LC-MS-based quantitative profiling was, therefore, optimized to measure 36 endogenous adrenal steroids and 3 therapeutic GCs of dexamethasone, prednisone, and prednisolone in human serum.  Enzymatic hydrolysis with -glucronidase and purification with solid-phase extraction were processed followed by LC-MS analysis.  In partial method validation, overall recovery ranged from 60.5% to 126.4%, except for 18-hydroxycorticosterone (18-OHB), in 0.1 to 500 ng/mL calibration ranges (r² ≥ 0.9137).  The precision (% CV) and accuracy (% bias) of the assay were 9.1% ~ 36.1% and 76.8% ~ 153.0%, respectively, with the LOQ of 0.1 ~ 5 ng/mL, except for 18-OHB.  The described technique would provide practical recommendations for the prevention and management of the complications of systemic GC theraphy and optimization for diagnosing cortisol excess by reducing false positive rate of DST.