Analysis of steroids in biological samples is used routinely in the diagnos
is of endocrine disorders. Binding assays (radioimmunoassays, immunosorbant
immunoassays and non-radioactive immunoassays) are reported often for the
analysis of single steroids in plasma and urine. Chromatographic methods th
igh-performance liquid chromatography and gas chromatography) are used for
steroid profiling where complex mixtures of steroids are analysed and the a
ctivity of biosynthetic and metabolic pathways deduced. Mass spectrometry i
s the ideal reference technique for detection of steroids, allowing high sp
ecificity and sensitivity. This review describes the practical issues conce
rning the quality of the assays performed and the potential pitfalls facing
the analyst in the design of such methods. Novel approaches for the quanti
fication of steroids, including microarrays and stable-isotope tracers are
described, with these being applied in the research environment as opposed
to routine biochemical laboratories.