This study was conducted to provide a more versatile and specific information on Thin Layer Chromatographic (TLC) analysis of medicinal plants. TLC plates developed with the extract of herbal medicines were analyzed with direct analysis in real time (DART®) ion source. Three well known herbal drugs were extracted and developed on a silica-coated TLC plate with the conditions pre-established in Korean Pharmacopoeia IX. The developed plate was placed between the DART® ion source and TOF-MS analyzer to get real time mass spectra from the bands on the TLC plate directly. The marker coumarin compounds, decursin and decursinol were successfully identified from the TLC plate developed with Angelicae gigantis radix, along with alkaloid compounds of rutaecarpine and evodiamine from Evodiae fructus, and lignan molecules of gomisin A, N, and schisandrin from Schisandrae fructus. This hyphenation system of TLC and DART®-MS could provide unique and specific information on the major constituents of crude plant drug on TLC through uncovering high resolution mass number of each band on the TLC plate directly in real time.