Abstract: Metabolites are small-molecule intermediates in metabolic pathways, which can interact with proteins and regulate protein functions in covalent or non-covalent ways. The interactions between metabolites and proteins play a crucial role in regulating cellular metabolism, influencing various biological processes, and contributing to disease development. Understanding these interactions is vital for elucidating metabolic pathways and designing therapeutic strategies for metabolic disorders and other diseases. However, studying metabolite-protein interactions presents several challenges due to the transient and dynamic nature of these interactions, as well as the vast diversity of metabolites and proteins in biological systems. Despite these obstacles, significant advances have been made in the past decade, particularly with the development of proteomics techniques, which have provided powerful tools to investigate these interactions with unprecedented depth and resolution. This review summarized the progress made in proteomics techniques for resolving protein-metabolite interactions over the past five years, including competitive active probes matched to target metabolites, direct capture methods based on chemical reactions, and label-free strategies for detecting endogenous protein-metabolite interactions. It further discussed the advantages, limitations, and application scenarios of these methods, aiming to provide a reference for studying protein-metabolite interactions. The competitive profiling strategy can covalently capture different types of metabolite-induced modifications with high selectivity and sensitivity. Significantly, this method has also been applied to profile modifications by reactive natural products or covalent drugs. On the other hand, direct capture methods also use chemoselective probes, but these probes are mostly applied in cell lysates due to their cell penetration and toxicity. Label-free strategies are able to detect non-covalent protein-metabolite interactions and record protein structural information in vitro, providing a more native, unmodified system for studying interactions of metabolites with proteins. However, extremely high sample reproducibility and extensive proteomic runs may not be feasible for regular labs. As a result, it is still challenging to uncover the complex nature of protein-metabolite interactions by label-free strategie. The development in analytical and bioinformatic approaches, as well as the advances in omics applications, offer new opportunities for this field. It is promising that a global landscape of the interaction proteins for the metabolites will be depicted in the future. This review provides insights into understanding the development of proteomic techniques for profiling protein-metabolite interactions.