Our understanding of biology has been greatly improved through recent developments in mass spectrometry, which is providing detailed information on protein and metabolite composition as well as protein-metabolite interactions. The high sensitivity and resolution of mass spectrometry achieved with liquid or gas chromatography allows for detection and quantification of hundreds to thousands of molecules in a single measurement. Where homogenization-based sample preparation and extraction methods result in a loss of spatial information, mass spectrometry imaging technologies provide the in situ distribution profiles of metabolites and proteins within tissues. Mass spectrometry—based analysis of metabolite abundance, protein-metabolite interactions, and spatial distribution of compounds facilitates the high-throughput screening of biochemical reactions, the reconstruction of metabolic networks, biomarker discovery, determination of tissue compositions, and functional annotation of both proteins and metabolites.