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Gas chromatography-mass spectrometry (GC-MS) has been used in hormone assays particularly for steroids in biological fluids. The combination of GC with MS exploits the high-resolving power of gas chromatography to separate closely related molecules, and the ability of the MS to provide precise data for identification and quantification of the separated substances. GC-MS is a very powerful technique for analysis with specificity of hormones in biological fluids. The general principles of GC-MS are described in this chapter along with some examples that illustrate specific applications of hormone analysis.