Mass spectrometry imaging (MSI) based on matrix-assisted laser desorption ionization (MALDI) is widely used in proteomics. However, matrix-free technologies are gaining popularity for detecting low molecular mass compounds. Small molecules were analyzed with nanostructured materials as ionization promoters, which produce low-to-no background signal, and facilitate enhanced specificity and sensitivity through functionalization. We investigated the fabrication and the use of black silicon and gold-coated black silicon substrates for surface-assisted laser desorption/ionization mass spectrometry imaging (SALDI-MSI) of animal tissues and human fingerprints. Black silicon was created using dry etching, while gold nanoparticles were deposited by sputtering. Both methods are safe for the user. Physicochemical characterization and MSI measurements revealed the optimal properties of the substrates for SALDI applications. The gold-coated black silicon worked considerably better than black silicon as the LDI-MSI substrate. The substrate was also compatible with imprinting, as a sample-simplification method that allows efficient transference of metabolites from the tissues to the substrate surface, without compound delocalization. Moreover, by modifying the surface with hydrophilic and hydrophobic groups, specific interactions were stimulated between surface and sample, leading to a selective analysis of molecules. Thus, our substrate facilitates targeted and/or untargeted in situ metabolomics studies for various fields such as clinical, environmental, forensics, and pharmaceutical research.