Nanomaterials have been widely used to assist laser desorption ionization of biomolecules for mass spectrometry analysis. Compared with classical matrix-assisted laser desorption ionization, strategies based on nanomaterial-assisted ionization generate a clean background, which is of great benefit for the qualitative and quantitative analysis of small biomolecules, such as therapeutic and diagnostic molecules. As label-free platforms, they have successfully been used for high-throughput enzyme activity/inhibition monitoring and also for tissue imaging to map in situ the distribution of peptides, metabolites and drugs. In addition to widely used porous silicon nanomaterials, gold nanoparticles can be easily chemically modified by thiol-containing compounds, opening novel interesting perspectives. Such functionalized nanoparticles have been used both as probes to extract target molecules and as matrices to assist laser desorption ionization for developing new enzyme immunoassays or for studying DNA hybridization. More recently, semiconductor nanomaterials or quantum dots acting as photosensitive centers to induce insource redox reactions for proteomics and to investigate biomolecule oxidation for metabolomics have been shown to offer new analytical strategies.