The characterization and quality control of natural herbal medicines, such as traditional Chinese medicines (TCMs), is of great significance to ensure their potential efficacy and avoid severe side effects. Thin-layer chromatography (TLC) is a simple and classic approach for examining quality marker of natural products. Nevertheless, it is more difficult to further characterize the compounds adsorbed on the TLC plate. Herein, we reported a simple setup of laser ablation-assisted direct analysis in real-time mass spectrometry (LA-DART-MS), in which the coupling of mass spectrometry information to provide a predominant dimension in the identification of unknown chemical compositions separated on standard TLC plates, and it was applied for rapid characterization of various kinds of natural herbal medicines. The results showed that the introduction of low-cost small laser pointer had significantly improved the desorption process. The system was successfully applied in the analysis of alkaloids, flavonoids, anthraquinones, volatile oils, glycosides, organic acids, and eight different TCMs including Sophorae Flavescentis Radix, Angelicae Sinensis Radix, Acori Tatarinowii Rhizoma, Phellodendri Chinensis Cortex, Picrasmae Ramulus et Folium, Gynura Japonica, Rhei Radix et Rhizoma and Dendrobii Caulis. The obtained limits of detection (LODs) of this method for various types of reference substances were in the range of 4.6–162.2 ng/band on TLC plates. Furthermore, the quality control and identification of different species of Dendrobii Caulis herb was achieved. This study combines the advantages of TLC and ambient mass spectrometry to provide a good choice for the screening and identification of active ingredients and the quality evaluation of botanical samples.
