The aim of this study was to develop a practical approach allowing a reliable and noninvasive assessment of cortisol production rates in premature infants. To measure daily urinary excretion rates of glucocorticoids, we developed a procedure using a hydraulic compression method to collect urine from cellulose nappies (diapers). Glucocorticoid metabolites were profiled by quantitative gas chromatography-mass spectrometry. Recovery of steroids after the process of hydraulic extraction from the nappy was approximately 100%. Consecutively, urinary excretion rates of glucocorticoids could be determined in nine healthy preterm infants. The median urinary excretion rate of glucocorticoids increased significantly during the first 5 d of life and remained between 566 microg/kg/d at d 5 and 302 microg/kg/d at 4 wk of age. However, this increase of urinary excretion rates of glucocorticoids in the first days of life was no longer significant when corrected for creatinine excretion. When calculated per square meter body surface area, the median urinary excretion rates of glucocorticoids were 5.1, 4.2, 4.1, and 3.7 mg/m2/d on d 5, and at wk, 2, 3, and 4, respectively. Urinary excretion rates of glucocorticoids constitute approximately 70% of the natural cortisol production rate as determined by stable isotope dilution technique in older children. Additionally, low cortisol production was detected in two of five preterm infants with arterial hypotension requiring treatment with catecholamines. In conclusion, 24-h urine collection using disposable nappies in combination with gas chromatography-mass spectrometry steroid profiling proved to be a reliable, noninvasive, nonstressful procedure to assess cortisol production and metabolism in premature infants.