Research directed at how phytases impact the fate of phytic acid (release of orthophosphate) in soil and sediments would be facilitated if an accurate/accessible measure of phytase activity were available. We are developing a soil phytase activity assay utilizing a novel chromophoric substrate analog of phytic acid, 5-O-[6-(benzoylamino)hexyl]-D-myo-inositol-1,2,3,4,6-pentakisphosphate, (T-IP5). The proposed soil phytase activity assay is based on measuring phytase-catalyzed dephosphorylation of T-IP5, in buffered soil slurry suspensions. We postulated on the reaction(s) that would take place under the assay conditions and have subsequently observed dephosphorylation of T-IP5 with subsequent production of T-inositol phosphate intermediates and T-myo-inositol (T-IP0): T-IP5 => T-IP4 => T-IP3 => T-IP2 => T-IP1 => T-IP0. Our T-IP5 probe allows for direct measurement of phytase-catalyzed dephosphorylation (i.e., hydrolysis of the phosphoester bond) using high-performance liquid chromatography with UV detection. Experiments were conducted to evaluate/define assay parameters including: (1) efficacy of using ethanol as a microbial growth inhibitor, (2) adsorption behavior of T-IP5 in the presences of common soil components (e.g. goethite), (3) probe stability (susceptibility of amide bond to hydrolysis), (4) effectiveness of extracting reagents on recovery of phosphorylated probe and (5) impact of citrate concentration on soil phytase activity. Results support our position that T-IP5 will function as a useful probe for measuring soil phytase activity.