Metabolites interact with proteins in vivo in various ways other than enzymatic reactions. Profiling of such interactions may help disclose unknown molecular mechanisms that regulate protein functions, and provide potential targets for disease treatment. Here we describe a procedure for systematic analyses of metabolite-protein interactions in vivo. This procedure couples protein affinity purification and mass spectrometry to identify metabolite-protein interactions. The primary effort can be completed within one day and scaled to process hundreds of samples in a batch. Originally developed in yeast, the same principle and protocol can be adapted to other organisms.