Introduction Marbofloxacin is a broad spectrum antibacterial agent which is very effective against a broad range of bacterial pathogens. The pharmacokinetic study of marbofloxacin is not conducted up till now in domestic ruminant species. So, there is a need to conduct pharmacokinetic study of marbofloxacin in order to select optimal dosage regimen in indigenous species. Objectives The study was conducted to compare inter and intra species variations of pharmacokinetic parameters and to select the dosage regimen of marbofloxacin in domestic ruminant species. Study Design Experimental study Setting Department of Pharmacology, Al-Nafees Medical College/Isra University, Islamabad Campus, Islamabad and Livestock Experimental Farm, Institute of Nutrition, University of Agriculture, Faisalabad. Duration July 2012 to December 2015. vii Material & Methods Thirty two adult, healthy, non-lactating females, animals were selected. 8 from each species of buffaloes, cows, sheep and goats of local breed. Marbofloxacin was injected as a single intravenous injection at a dose of 2.5 mg/kg body weight to each animal. Blood and urine samples were collected at specific time intervals. The concentration of marbofloxacin in blood and urine were determined by HPLC method. Data were subjected to computer software WinNonlin® to obtain pharmacokinetic parameters. Inter and intra species differences were observed by ANOVA followed by Duncan’s Multiple Range Test. . Results The low values of t1/2a suggested the rapid distribution of marbofloxacin in all four species. The values for the elimination half life (t1/2b) were insignificant to each other in all tested species. The volume of distribution at terminal phase (Vdarea) and at steady state (Vss) was comparable (p>0.05) and higher (p<0.05) in sheep and goats followed in descending order in cows and buffaloes indicating the well distribution and deep tissue penetration of drug. However, the mean values for Vc were remarkably higher in sheep (2.5±0.14 L/kg) followed by goats (2.21±0.11 L/kg) and parallel values in buffaloes (0.76±0.013 L/kg) and cows (0.88±0.02 L/kg). Total body clearance (ClB) was expressed as L/h/kg, and it showed the similar pattern to Vdarea and Vss in local ruminants. The value of AUC was viii significantly (p<0.05) higher in buffaloes (8.37±0.50 μg.h/mL) followed by cows (4.46±0.30 μg.h/mL), and parallel (p>0.05) values in sheep (2.08±0.05 μg.h/mL) and goats (2.16±0.04 μg.h/mL). In vitro the values of plasma protein binding of marbofloxacin were 32.2%, 27.01%, 20.15% and 21.17% in buffaloes, cows, sheep and goats respectively. The MIC50 and MIC90 values for marbofloxacin against Staphylococcus aureus species were observed to be 0.5 and 1.0μg/ml respectively with MIC range of 0.125-2.0 μg/mL. Endogenous creatinine was used as an index of glomerular filtration rate (GFR) and recorded to be 0.87 ± 0.06 mL/min/kg in buffaloes, 0.98 ± 0.05 mL/min/kg in cows, 1.29 ± 0.079 mL/min/kg in sheep and 1.49 ± 0.103 mL/min/kg in goats. The renal clearance of marbofloxacin was measured as 1.96 ± 0.51 mL/min/kg in buffaloes, 2.08 ± 0.46 mL/min/kg in cows, 1.83 ± 0.54 mL/min/kg in sheep, 2.15 ± 0.53 mL/min/kg in goats. In the renal handling of marbofloxacin, glomerular filtration and tubular secretion were involved in all ruminant species. Conclusion The dosage regimen of marbofloxacin was higher in domestic ruminants as compared to its foreign counterparts. Keywords Marbofloxacin, Domestic Animals, Pharmacokinetics, Renal Clearanc, HPLC.