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Aptamers are efficient multifunctional binding molecules that can recognize various types of target molecules. Even challenging targets, such as small molecules, can be recognized and bound by aptamers. However, due to the lack of mature analytical methods, it is difficult to study the interactions between aptamers and drugs, antibiotics or metabolites in detail. Fundamental binding parameters of these small molecule-aptamer interactions, such as binding affinity, stoichiometry, and thermodynamics, were carefully obtained using state-of-the-art techniques. The innovative microscale thermophoresis (MST) is a novel, rapid and precise method to characterize these small molecule-aptamer interactions in solutions at the microliter scale.