Traditional Chinese medicines (TCMs) are attracting more and more attention because of their long historical clinical experience and reliable therapeutic efficacy for preventing and/or treating various human diseases. Many techniques and methods were developed for the analysis of TCMs to support new drug discovery and quality control. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), a soft ionization mass spectrometric technique, has been widely used in the analysis of a wide variety of large molecular compounds including proteins, peptides, and polymers since it was introduced in the late 1980s. In the present chapter, advances of MALDI-TOF MS in TCMs analysis have been reviewed. The review covers MALDI-TOF MS applications in the identification of new bioactive ingredients, analysis of alkaloids, determination of small molecular compounds with new matrices, proteomics analysis associated with TCMs, direct analysis of plant tissue, and other applications in TCMs.