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The analysis of interactions between biological agents or with surrounding chemicals is important in many areas of modern biochemical, biomedical, and environmental research. Microscale platforms based on affinity chromatography have been shown to be a powerful set of tools for these studies. This approach makes use of an immobilized binding agent as a stationary phase in a microscale platform for either direct examination of the interactions of this agent with an applied target solute or as a secondary capture agent to probe a solution‐phase interaction. This review will examine the various platforms and strategies that have been used in microscale affinity chromatography, or µAC, to characterize and study biointeractions. The general principles of µAC and schemes based on this approach will be examined, along with applications of this technique. Examples of approaches that will be considered will include zonal and frontal analysis methods, as well as a variety of schemes by which µAC can be employed in kinetic studies. In each case, the theory and principles of these methods will be provided along with examples of their use in biointeraction studies.