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A two-period cross-over study was carried to investigate the pharmacokinetics (PK) and ex-vivo pharmacodynamics (PD) of cefquinome when administrated intravenously (IV) and intramuscularly (IM) in seven healthy dogs at a dose of 2 mg/kg of body weight. Serum concentrations were determined by HPLC-MS/MS assay and cefquinome concentration vs. time data after IV and IM were best fit to a two-compartment open model. Cefquinome mean values of area under concentration–time curve (AUC) were 5.15 μg·h/mL for IV dose and 4.59 μg·h/mL for IM dose. Distribution half-lives and elimination half-lives after IV dose and IM dose were 0.27 and 0.44 h, 1.53 and 1.94 h, respectively. Values of total body clearance (ClB) and volume of distribution at steady-state (Vss) were 0.49 L·kg/h and 0.81 L/kg, respectively. After IM dose, Cmax was 2.53 μg/mL and the bioavailability was 89.13%. For PD profile, the determined MIC and MBC values against K. pneumonia were 0.030 and 0.060 μg/mL in MHB and 0.032 and 0.064 μg/mL in serum. The ex vivo time-kill curves also were established in serum. In conjunction with the data on MIC, MBC values and the ex vivo bactericidal activity in serum, the present results allowed prediction that a single cefquinome dosage of 2 mg/kg may be effective in dogs against K. pneumonia infection.