An introduction to conjugated steroids and the justification for their analysis is provided covering both environmental and biological samples. Determining conjugated steroids or indeed any organic chemical which is conjugated upon excretion from the body has relevance in diagnostic monitoring, forensic screening and environmental analysis (from the endocrine disrupter perspective). The various analytical approaches and the accompanying issues are application-dependent. There are numerous options at each stage of analysis, from extraction, hydrolysis, derivatisation, and detection, and advances can be confined to the specific application for which it was developed. Emphasis is placed on the choice of separation and how gas or liquid chromatography necessitates different preparative stages to enable conjugated steroid determination. Possible future directions and research for conjugated steroid analysis are discussed.