The role of EDTA in tissue penetration was studied in present investigation. The present study has been designed to determine the most appropriate concentration of EDTA in FDC of ceftriaxone and sulbactam (Sulbactomax; 500 mg) in various tissues with three different concentration of EDTA. Total 18 rats were divided into three groups. Group I (n=6) treated with sulbactomax drug + EDTA (0.37mg/ml); group II (n=6) treated with sulbactomax + EDTA (3.7mg/ml) and group III (n=6) treated with sulbactomax + EDTA (37.0 mg/ml). The drug was analyzed by HPLC in various tissues. Our results showed that the concentration of ceftriaxone was statistically significantly (p<0.001) increased in kidney, lung and stomach tissues. Similarly, concentration of sulbactam was also found to be increased in kidney, liver and stomach tissues of group II as compared to the group I and III. The penetration of both drug was increased in group II of various tissues due to EDTA in comparison to ceftriaxone alone (without EDTA). EDTA competes with microorganism for any of the trace metal ions that are essential to the maintenance of their life cycle or it disturbs the integrity of cell membrane. Our finding concluded that sulbactomax combined with EDTA (3.7 mg/ml) has better tissue penetration in vitro and hence improves efficacy that might be effective against a variety of infections.