Methods for the detection of specific interactions between diverse proteins and various small-molecule ligands are of significant importance in understanding the mechanisms of many critical physiological processes of organisms. The techniques also represent a major avenue to drug screening, molecular diagnostics, and public safety monitoring. Terminal protection assay of small molecule-linked DNA is a demonstrated novel methodology which has exhibited great potential for the development of simple, sensitive, specific and high-throughput methods for the detection of small molecule-protein interactions. Herein, we review the basic principle of terminal protection assay, the development of associated methods, and the signal amplification strategies adopted for performance improving in small molecule-protein interaction assay.