Objective: The aim is to determine cortisol concentration in the 24-hour urine sample of patients with a referral diagnosis of adrenal cortex disorders, to interpret obtained results according to the pathophysiological characteristics of certain adrenal gland disorders and to describe the importance of determining cortisol in a 24-hour urine sample as one of the diagnostic parameters for establishing a diagnosis and monitoring the course of the disease for patients with adrenal gland disorder. Materials and methods: 10 patients of UHC Split were selected with a referral diagnosis that included some of the adrenal cortex disorders and a request to determine the concentration of cortisol in 24-hour urine. Patient samples were processed and analyzed at the Department of Medical Laboratory Diagnostic according to the protocol of the manufacturer Roche "Elecsys Cortisol", using the automated ECLIA method for urinary free cortisol on the Cobas 6000 analyzer. Results: The reference interval for this method defined by manufacturer is 100 – 379 nmol/dU. Four patients had a cortisol concentration below 100 nmol/dU, five patients above 379 nmol/dU, and one patient in the range 100 – 379 nmol/dU. The results were observed in the context of the referral diagnoses of the patients. Conclusion: The cortisol concentration in urine is not a sufficient parameter to make a diagnosis, but it directs further diagnostic procedure and brings the doctor closer to the answer. The accuracy and precision of the method, correct sample processing and handling as well as proper sample are essential for correct results.