Catecholamines and steroids are well-known neurotransmitters and hormones that rapidly change the excitability of neurons. Alopecia areata is a disease for which the exact cause is unknown, but it is considered to be associated with stress, and so the simultaneous analysis of catecholamines and steroids is required for the diagnosis of alopecia areata. Thus, we herein report the simultaneous analysis of catecholamines and steroids bearing different functional groups for the first time, during which it was necessary to carry out a serial hydrolysis procedure. Following hydrolysis of the urine samples to produce the free forms from the urinary conjugates, ethyl acetate extractions were carried out, and chemical derivatization was performed using dansyl chloride to increase the sensitivity of the liquid chromatography–tandem mass spectrometry method. The matrix effects and recoveries of this analytical method were validated, giving values of 85.4–122.9% and 88.8–123.0%, respectively. In addition, the method accuracy and precision were assessed, giving values of 0.4–21.5% and 2.0–21.6% for the intra-day and inter-day precisions, respectively. This validated method was then applied to identify differences between patients with and without alopecia areata, wherein the metanephrine content was found to be significantly higher in the alopecia areata patient group. This quantitative profiling method can also be applied to steroid-dependent diseases, as well as catecholamine-related diseases.