Bacteria rely on signal transduction pathways to respond to environmental changes. Many of these pathways are two-component systems (TCSs). The prototypical TCS includes two proteins that are often encoded on the same operon and have molecular recognition for one another: a membrane-bound histidine kinase (HK) and a cytosolic response regulator (RR). Bacterial HKs are typically homodimeric, periplasmic sensing proteins anchored to the membrane. The most direct way of detecting TCS activity is by monitoring the phosphorylation of its proteins, for which radioactive adenosine triphosphate (ATP) assays have traditionally been used. The ideal use of an ABP will be the global profiling of HKs from various sample types or screening of inhibitors. HKs of TCSs are excellent drug targets as it is well established that HKs aid in bacterial survival and virulence. Preventing these signaling pathways from transmitting information would be a new way of treating bacterial infections.