The subtypes of congenital adrenal hyperplasia (CAH) are distinguished by defects in enzymes responsible for aberrant steroidogenesis, particularly in cortisol production. Traditional diagnosis of CAH typically involves a labor-intensive process and has limitations in distinguishing between subtypes. Through LC-MS-based steroid profiling, a single analytical run reveals substantially elevated serum levels of specific steroids and metabolic ratios corresponding to the enzymatic activities of defective steroidogenic enzymes in the 21-OHD, 17α-OHD, and 11β-OHD subtypes of CAH.