A novel plasma assisted multiwavelength (1064, 532, and 355 nm) laser desorption ionization mass spectrometry (PAMLDI-MS) system was fabricated and applied in the analysis of low molecular weight compounds through combination with thin layer chromatography (TLC). The TLC/PAMLDI-MS system successfully integrated TLC, the multiwavelength laser ablation, and the excitated state plasma from direct analysis in real time (DART®) and was proved to be effective in the facile separation and selective identification of low molecular weight compounds. An automated three-dimensional platform was utilized to facilitate the analysis procedures with all the parameters of the TLC/PAMLDI-MS systematically optimized, and the desorption/ionization mechanisms were discussed. The successful combination of three-wavelength laser with DART® based system extended the range of the analytes and provided broad possibilities for the compound desorption from the TLC. The experimental results clearly showed that the laser desorption was wavelength dependent. The PAMLDI-MS system was successfully applied in the detection of low molecular weight compounds from different kinds of samples separated on a normal-phase silica gel, such as dye mixtures, drug standards, and tea extract, with the detection level of 5 ng/mm(2).