Two experiments were performed for evaluating calibration curve (CC) and comparing negative and positive controls (CNP) as a major method for estimating of phytase phosphorus equivalence for layer and broiler chickens. In the first and second experiments, 360 Hy-line W-36 layer hens and 525 day-old Ross-308 broiler chickens were used in a complete randomized design, respectively. Evaluated methods were setting the two regression equations for NPP-supplemented and phytase supplemented treatments with two sub-methods, include calibration curve (CC) or exclude the amount of phosphorus content of basal diet (CC-BD) in calculation, and exploring enzyme equivalency by comparing phosphorus deficient diet as an negative and supplemented diet by inorganic phosphorus sources as a positive control group (CNP). Experiment one included nine treatments (200, 300, 400 and 500 FTU/kg phytase was added to a phosphorus deficient basal diet contained 0.12% Av.P, the rest four treatments were included basal diet supplemented with 0.20, 0.27, 0.35 and 0.43% Av.P). Experiment two included seven treatments (a basal P deficient diet contained 0.27% Av.P, and two increasing levels of Av.P, 0.32 and 0.37%, and four doses of phytase 200, 300, 400 and 500 FTU/kg added to basal diet). Each treatment in the both experiments replicated five times. Results indicated that methods of estimation had a significant effect on phosphorus equivalence estimation (P<0.0001). Fitted regression equations considering P content of basal diet (CC-BD) estimated rational values than those ignore it (CC) (0.161% vs 0.424% and 0.432% vs 0.564% for 500 FTU/kg phytase for broiler chicken and layer hens, respectively) (P<0.0001). On average, among three methods used, CC method had the highest estimated values both in broiler chickens and layer hens (p<0.0001). Regardless of mathematical method, there were different significant values for different strains (layer, 0.381% and broiler, 0.212%) (P<0.0001), but not for different traits served as response criteria (P>0.05). In conclusion, the phosphorus equivalent value of enzyme varies according to the estimation methods and strain. Hence, using matrix values of enzyme for accurate feed formulation depend on a variety of circumstances and decision making requires comprehensive information.