Nanomaterials, primarily nanoparticles (NPs), can serve as an alternative matrix for the analysis of various biomolecules through SALDI MS. SALDI MS has been developed to overcome poor reproducibility and high background in the low mass region commonly occurring in MALDI MS. Various nanomaterials, unmodified or functionalized with recognition ligands, can have a strong affinity toward certain analytes and thus are applicable for their concentration and enrichment from complex biological matrices. In mass spectrometry imaging (MSI), the use of NPs instead of the conventional matrices can improve the spatial resolution up to the cellular level. In this review, the nature of NPs, the methods of sample preparation and approaches for quantitation of biomolecules through SALDI MS are discussed. Practical applications and limitations of SALDI MS employing NPs for separate samples and MSI are mentioned. With regard to the nature of MSI analysis, the use of nanostructured surfaces for MSI is also reflected in this review.