The method described in this chapter provides a quantitative means of assaying for protein histidine phosphorylation and thus protein histidine kinase activity, even in the presence of other protein kinases, for example, serine/threonine or tyrosine kinases. The method involves the measurement of 32P, derived from [γ32P]ATP, incorporation into phosphohistidine in a protein substrate. The method makes use of the differential stabilities of phosphohistidine and the common phosphohydroxyamino acids to alkali and acid treatments to measure phosphohistidine incorporation. Phosphoserine and phosphothreonine are depleted by alkali treatment, while phosphohistidine, which is alkali-stable, is removed by acid treatment. Phosphotyrosine is stable to both alkali and acid treatments. The method is filter-based and allows for rapid assay of multiple protein histidine kinase samples, for example, screening for histidine kinase activity, allowing for the calculation of specific activity. In addition, quantitative time-course assays can also be performed to allow for kinetic analysis of histidine kinase activity.