Two-component systems of bacteria play many crucial roles in antibiotic resistance, virulence, and biofilm regulation. Accordingly, TCS inhibitors have been investigated as attractive antibacterial drug candidates. Current HK activity assays are hindered by the notorious instability of the phosphohistidine (pHis) product. To overcome these difficulties, we developed a simple and high-throughput screening method named AUDECY (AUtophosphorylation-DEphosphorylation CYcle assay). This convenient kinetic assay successfully measured the enzyme activity of multiple HKs, especially those with very low pHis levels, thereby overcoming the limitation of current endpoint HK assays. Through a high-throughput screening with this method, we also identified a potent VanS HK inhibitor, which sensitized vancomycin-resistant Enterococcus faecium (VRE) toward vancomycin, highlighting the potential of this assay in HK inhibitor discovery.