An innovative approach for measuring phytase activity (PA) in surface water is presented. A substrate analog of myo-inositol hexakis(dihydrogen) phosphate (InsP6), commonly referred to as phytic acid, 1d-myo-5-O-(1-oxo-1-(2′ ,4,7,7′ -tetrachloro-3′,6′-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9’-xanthen]-6-yl)-5,8,11-trioxa-2-azatridecan-13-yl)-inositol 1,2,3,4,6-pentakis-O-(dihydrogen) phosphate, referred to as tetrachlorofluorescein (TET) tethered (T)InsP5, has been developed that can be used to monitor the (phytase-catalyzed) phosphate ester bond–cleavage reaction. Test phytases, (wheat [4-] and Aspergillus niger [3-] phytase) sequentially remove phosphate groups from TET TInsP5, producing dephosphorylated probe species that were readily separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Because dephosphorylated probe species retain the TET group, highly sensitive quantification could be achieved using fluorescence detection (excitation/emission ′ = 245/540 nm). Calibration curves for TET TInsP5, which could be used as a standard for quantifying all probe species, were linear (R2 > 0.999) over the range of concentrations tested. Phytase-generated dephosphorylated probe species were characterized or identified using RP-HPLC with mass spectrometry. Results of mass spectrometry analysis show that the RP-HPLC system was capable of distinguishing between dephosphorylated probe species at the regioisomeric level. The TET TInsP5 molecular probe was used to successfully measure PA in pond water. We found that the PA associated with the particulate plus water-soluble fraction was greater than that observed for the water-soluble fraction alone. Moreover, it appeared that 4- and 3-phytase were active in pond water based on an analysis of the chromatographic profile (i.e., elution sequence) of dephosphorylated probe species produced. The advent of a fluorescent substrate analog of InsP6 affords environmental scientists with the means to unambiguously quantify an extremely small amount of phytase-generated dephosphorylated product(s), enabling the measurement of PA over a reasonably short time duration, in an environmental sample containing low concentrations of enzyme.