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Urinary steroid profiling provides quantitative information on the steroid biosynthetic and catabolic pathways. It is essential for identification of inborn errors of steroid metabolism, and useful in other disorders with altered steroid secretion. A general method is detailed. Steroids, mostly in the form of glucuronide and sulphate conjugates, are extracted using solid-phase cartridges, followed by enzymatic hydrolysis, reextraction of freed steroids, formation of methoxime trimethylsilyl derivatives and analysis by sas chromatography and gas chromatography-mass spectrometry. Newborns excrete large quantities of sulphates, so conjugate separation by liquid gel chromatography is used prior to hyrolysis. Normal ranges for adults and children are given, together with advice on chromatogram evaluation and a summary of the profile findings in steroid disorders.