The interactions of drugs with serum proteins are often stereoselective and can affect the distribution, activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have been used as tools to study these interactions. This review describes the general principles of affinity chromatography and HPAC as related to their use in drug binding studies. The types of serum agents that have been examined with these methods are also discussed, including human serum albumin, α1-acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on affinity chromatography and HPAC that have been used to investigate drug interactions with serum proteins and the historical development for each of these formats. Specific techniques that are discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use of band-broadening measurements, peak decay analysis, or ultrafast affinity extraction.