A chip-based platform for laser desorption/ionization mass spectrometry (LDI-MS) analysis of small molecules was developed by utilizing layer-by-layer (LBL) assembly of MoS2 nanoflakes and polyallylamine on an arbitrary substrate. The LDI-MS efficiency of small molecules on MoS2 films increased as a function of LBL assembly cycles until reaching a saturation point. The optimized MoS2 nanoflake film exhibits high LDI-MS efficiency, salt tolerance, reusability and uniform ionic signal distribution, and its performance was further enhanced by surface modification with perfluoroalkanes mimicking a clathrate nanostructure.