Thin-layer chromatography (TLC) is a widely used, fast and inexpensive method for separating complex mixtures. Unfortunately, the quality of achievable separation represents only one side. An additional problem is the unambiguous assignment of the obtained spots to defined compounds. Clear identification of spots is often not possible by common staining methods and comparison with a known reference compound. Therefore, further analytical techniques are mostly required for further structural elucidation. Mass spectrometry (MS) is a suitable method due to its high sensitivity. In particular, matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) MS is a modern soft-ionization technique that may be easily combined with TLC. This review summarizes the so far available knowledge about direct TLC–MALDI combination and gives an overview about different molecule classes that have already been successfully analyzed by this approach. This review critically summarizes the capabilities and limitations of the direct MALDI–TLC combination and highlights in particular the problems related to sample preparation and instrumentation.