Thin-layer chromatography (TLC) is a widespread technique because it allows fast, simple, and inexpensive analyte separations. In addition, direct analysis of the compounds separated on TLC plates via mass spectrometry (MS) has been shown to provide high sensitivity and selectivity while avoiding time-consuming sample extraction protocols. Here, direct desorption low-temperature plasma-mass spectrometry (LTP-MS) as well as diode laser assisted desorption (LD) LTP-MS are studied for direct spatially resolved analysis of compounds from TLC plates. Qualitative and quantitative characterization of amino acids, pharmaceuticals, and structural isomers were performed. The nature of the TLC plate stationary phase was found to have a significant influence, together with the analyte's characteristics, on the desorption efficiency. Tandem MS is shown to greatly improve the limits of detection (LODs). Direct desorption LTP-MS, without external thermal assisted desorption, demonstrates its best performance with cellulose TLC plates (LODs, 0.01 ng/mm 2 to 2.55 ng/mm 2 ) and restricted performance with normal-phase (NP) TLC plates (several analytes without observable signal). LD LTP-MS, with systematic optimization of irradiance and focal point diameter, is shown to overcome the direct-desorption limitations and reach significantly improved LODs with NP TLC plates (up to ×1000 better). In addition, a wide-ranging characterization of amino acid analytical figures of merit with LD LTP-MS shows that LODs from 84 pg/mm 2 down to 0.3 pg/mm 2 are achieved on NP TLC plates.