Abstract: Enzymes play crucial functional roles in all biological processes. Enzymatic inhibitors can regulate enzyme activity and may become the starting point for drug discovery. Mass spectrometry (MS) has the advantage for rapid qualitative and quantitative analyses of compounds and enzyme reactions, emerging as an important analytical tool in enzyme inhibitor screening assay for drug discovery. This review will highlight recent advances in the inhibitor screening assay using MS and related techniques, including frontier affinity chromatography, immobilized enzyme beads, ultrafiltration, surface plasmon resonance, capillary electrophoresis and microfluidic chips. The existing MS methods for screening enzyme inhibitor were divided into two types:affinity screening and activity screening.