While two-component systems (TCSs), composed of a sensor histidine kinase (HK) and a response regulator, are the main signaling pathways in bacteria, global TCS activity remains poorly described. Here, we report the kinetic parameters of the HK autophosphorylation reaction using previously uncharacterized γ-phosphate-modified ATP analogues to further elucidate their utility as activity-based probes for global TCS analysis. Given the increased stability of thiophosphorylated histidine in comparison to that of the native phosphoryl modification, which is attributed to the decreased electrophilicity of this moiety, we anticipated that ATPγS may be turned over much more slowly by the HKs. Surprisingly, we found this not to be the case, with the turnover numbers decreasing <1 order of magnitude. Instead, we found that alkylation of the thiophosphate had a much more dramatic effect on turnover and, in one case, the binding affinity of this substrate analogue (BODIPY-FL-ATPγS).