Polystyrene (PS) is a class of polymer materials that offers great potential for various applications. However, the applications of PS microspheres in paper spray mass spectrometry are largely underexplored. Herein we prepared a series of PS microspheres via a simple dispersion polymerization and then used them as coating materials for paper spray mass spectrometry (MS) in high-sensitivity analysis of various therapeutic drugs in complex biological matrixes. In the preparation of PS-coated papers, the coating method was found playing a key role in determining the performance of the resulting paper substrate in addition to other parameters (e.g., starch type and amount, PS coating amount, and spray solvent). We also found that as a solvent was applied on PS-coated paper for paper spray, the analytes of interest would be first extracted out and then moved to the tip of paper triangle for spray along with the applied solvent. In the process, the surface energy of PS particles had a strong impact on the desorption performance of analytes from PS-coated paper substrate, and the PS with a high surface energy favored the elution of analytes to allow a high MS sensitivity. When the prepared PS coated paper was used as a substrate for paper spray, it gave high sensitivity in analysis of therapeutic drugs in various biological matrixes such as whole blood, serum, and urine with excellent repeatability and reproducibility. In contrast to uncoated filter paper, an improvement of 10-546-fold in sensitivity was achieved using PS-coated paper for paper spray, and an estimated lower limit of quantitation (LLOQs) in the range of 0.004-0.084 ng mL-1 was obtained. The present study is significant in exploring the potential of PS for high-sensitivity MS analysis, and it provides a promising platform in the translation of the MS technique to clinical applications.