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This chapter describes how adrenocorticotropin (ACTH) can be measured in biological fluids. The principle can be applied to similar peptide hormones. Following an introduction of the principle of the method, details are given on sample collection and storage, and materials required. Detailed descriptions are given for the methodology with a “Notes” section that gives practical tips on the more complex or difficult steps in the analytical process. A brief bibliography lists peer reviewed articles relevant to the development and application of this method.