Post-translational modifications (PTMs) play important roles in regulating the target proteins’ functions and physiological outcomes. Phosphorylation is an example of PTMs crucial in cell signaling and metabolism. For example, histidine phosphorylation is found in two component system (TCS), the most popular bacterial signaling system, which functions through the phosphorylation of histidine and aspartate residues. Histidine phosphorylation is also implicated in chromatin remodeling via the phosphorylation of histone H4. However, histidine phosphorylation has not been thoroughly investigated while phosphorylation on serine, threonine and tyrosine has been studied well, because of phosphohistidine’s chemical instability and the resultant deficiency of proper research techniques and tools. To address this, we report our progress to develop fluorescence-based chemosensors for histidine phosphorylation. Herein, we report our progress to develop fluorescence-based chemosensors for histidine phosphorylation. Preliminary results in the application to real-time assays of histidine kinases and phosphatases are also presented