Development of fluorescence polarization (FP) assays, especially in a competitive manner, is a potent and mature tool for measuring the binding affinities of small molecules. This approach is suitable for high-throughput screening (HTS) for initial ligands and is also applicable for further study of the structure-activity relationships (SARs) of candidate compounds for drug discovery. Buffer and tracer, especially rational design of the tracer, play a vital role in an FP assay system. In this perspective, we provided different kinds of approaches for tracer design based on successful cases in recent years. We classified these tracers by different types of ligands in tracers, including peptide, nucleic acid, natural product, and small molecule. To make this technology accessible for more targets, we briefly described the basic theory and workflow, followed by highlighting the design and application of typical FP tracers from a perspective of medicinal chemistry.