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Phytate (inositol hexaphosphate) has been identified as a major organic phosphorus (P) form in soil, animal manure, and other environmental samples. Although a number of methods are available for quantitative isolation and determination of phytate, they are time-consuming and not amenable to routine analysis. We developed a simple, rapid method for enzymatic determination of phytate in animal manure. Animal manure was extracted by H2O, 1M hydrochloric acid (HCl), 0.1M sodium acetate (NaOAc, pH5.0) with or without 0.05M ethylenediaminetetraacetate (EDTA), and 0.25M or 0.5M sodium hydroxide (NaOH)–0.05M EDTA. Extracts were diluted (1/10–1/150) and adjusted to pH5.0 in sodium acetate buffer. The diluted extracts were then incubated at 37 °C for 1 h in the absence and presence of fungal 3-phytase (PHY) and potato acid phosphatase (PAP). Enzymatic hydrolyzable organic P was calculated as the difference in inorganic P (Pi) between the mixtures with and without enzymes. Our data indicated that enzymatic incubation of properly diluted and pH-adjusted HCl or NaOH/EDTA extracts released phytate P. The complementary substrate specificity of the two enzymes is considered to enhance the effectiveness of enzymatic hydrolysis. Consequently, we recommend this method of combining PAP and PHY for quantifying phytate P. Additional research is being conducted to verify the effectiveness of this method for general use across a wider range of soils and manures.