The bioavailability of ofloxacin, a fluoroquinolone widely used in the treatment of bacterial infection varies different with different binders used in the formulation of tablets due to different binding properties and variable release characteristics. In this study, two formulations of ofloxacin were prepared. The only difference between them was of binder. The two binders used were gelatin and starch. In-vitro and in-vivo evaluation of tablets was performed. Eight healthy human volunteers were selected for this study, and were divided into two groups each consisting of 4 volunteers. First group was given formulation 1 with gelatin as binder. Each volunteer received 200 mg ofloxacin tablet. Volunteers of the second group were given formulation 2 with starch as binder. After one week wash out period, volunteers of the first group received formulation 2 and volunteers of second group received formulation 1. Blood samples were collected at different time intervals. The drug concentrations in plasma were assayed by High Performance Liquid Chromatography. Pharmacokinetic parameters of formulation 1 were Cmax 1.4412  1.8367 µg/ml, tmax was 1.00 ± 0.00 hours, AUC 8.6804 ± 0.8346 µg.h/ml, AUMC 43.017 ± 0.2893 µg.h2/ml, MRT 4.8869±1.3587 hours, Ke 0.2067 ± 6.9207, T½ 3.3886 ± 1.6321 hours, Vd 113.826±0.2983 L/Kg, Vss 4.833 ± 0.9138 L/Kg, Cl 23.595 ± 0.5070 ml/h/Kg. For Formulation 2 these values were 1.515  1.5898 µg/ml, 0.5 ± 0.00 hours, 9.0317 ± 0.8805 µg.h/ml, 35.4486±0.3337 µg.h2/ml, 3.8798 ±1.4668 hours, 0.2606 ±6.0291, 2.68 ± 1.76 hours, 86.609 ±0.3354 L/Kg, 5.94 ±0.84L/Kg, 22.580 ±0.5333 ml/h/Kg respectively. Statistical analysis was performed and it was found that the formulation 1 (formulated with gelatin) released the drug slightly greater than the formulation 2 within two hours after its administration. There was highly significant difference between mean residence time, elimination rate constant, half life and volume of distribution between both of the formulations. Therefore, formulation 2 has greater bioavailability than the formulation 1. Thus it can be concluded that the binder can affect the bioavailability and pharmacokinetic parameters of a drug.