Inorganic phosphate (Pi) is one of the most important ions in biological systems. It functions in a variety of roles. One of the most important roles is as a molecular switch, turning enzyme activity on and off through the mediation of the various protein kinases and phosphatases in biological systems. A highly sensitive assay is desired to monitor Pi in variety samples or monitor Pi changes during kinase and phosphatase reactions. The newly designed Phosphate Assay Kit provides a sensitive, easy, quick means of assessing phosphate over a wide range of concentrations. In the assay, inorganic phosphate reacted with maltose to produce glucose in the present of a special enzyme. The glucose is specifically oxidized to generate a product that reacts with the OxiRed® probe to generate fluorescence (Ex/Em=535/587nm). The kit can be used to detect Pi produced through reactions involving ATPases, GTPases, 5’-nucleotidase, protein phosphatases, acid and alkaline phosphatases, and phosphorylase, etc. from a variety of samples. Glucose background can be eliminated by doing a glucose control. Phosphate concentrations between 0.4 µM and 10 µM, with a lower detection limit of approximately 40 pmol can be directly determined.