A variety of analytical methods are used in today’s clinical laboratory, but one of the most common is that of high-performance liquid chromatography (HPLC). Although the majority of clinical work in HPLC is performed with traditional methods such as reversed-phase, size-exclusion, and ion-exchange chromatography, another approach that is gaining in importance is the technique of affinity chromatography. In this chapter the general principles of this method will be examined and the basic components of an affinity chromatographic system will be described. A variety of clinical and pharmaceutical applications of this technique will also be presented, with an emphasis being given to HPLC-based methods for chemical analysis and quantitation. These applications will include traditional affinity methods as well as systems in which affinity columns are used in automated immunoassays or in combination with other techniques to perform on-line extraction or post-column detection.