Zinc (Zn) is the most vulnerable cation to complexation with phytate. An experiment was conducted to evaluate the potential of measurements of Zn concentration in the gastrointestinal tract as a marker to assess the anti-nutritional impact of phytate and susceptibility of phytate to phytase in broilers. Ross 308 broilers (n = 180) were fed one of 5 experimental diets with differing phytase activity levels, analyzed at 605, 1150, 1804, 3954, and 5925 U/kg. Broiler performance and Zn concentration, pH, and amount of phytate hydrolyzed in the gizzard, duodenum, and ileum were analyzed at d 21 post hatch. Phytate susceptibility to phytase degradation was determined in vivo and in vitro by measuring total phytate-P hydrolyzed in the tract or in conditions that mimicked the tract, respectively. Phytase activity level had a significant (P < 0.05) impact on Zn concentration and phytate hydrolyzed in the gizzard and ileum, but not in the duodenum. Strong relationships were observed between the amount of phytate hydrolyzed and Zn concentration in the gizzard in birds fed the diets with 1804 U/kg or higher levels of phytase. Phytate and phytase effects could therefore potentially be evaluated by measuring Zn concentration in the gizzard. Susceptible phytate levels measured in vivo and in vitro were almost identical in the diet with phytase activity of 5925 U/kg, but in the diets with lower phytase activity levels the in vitro assay overestimated the amount of P released. There were strong relationships between in vivo susceptible phytate level and pH and amount of phytate hydrolyzed in the gizzard, duodenum, and ileum and Zn concentration in the gizzard and ileum. This illustrates that phytate susceptibility directly effects mineral availability in the gastrointestinal tract. Measurements of Zn concentration in the gastrointestinal tract, particularly in the gizzard, can potentially be used as a marker to assess the anti-nutritional impact of phytate and susceptibility of phytate to phytase in broilers.