Ion exchange chromatography (IXC) has been utilized in many different ways in the field of steroid analysis already for more than thirty years. Steroid conjugates can be extracted and fractionated on anion exchangers by virtue of their anionic nature, and in the same way the weakly acidic phenolic steroids are separated from the neutral ones. Anionic and cationic contaminants are removed by IXC during purification of the samples, and further, the use of lipophilic ion exchangers allow the simultaneous reversed phase chromatographic elimination of neutral lipids. Finally, certain steroids with specific functional groups can be selectively fractionated by ion exchangers converted to a suitable form. The high capacity and flexibility of IXC has made it a technique of choice in the purification of biological samples for the multicomponent analysis of steroids by GC or GC-MS during the last decade.