The aim of this study was to evaluate the pharmacokinetics and bioavailability of cefquinome (CFQ) and ceftriaxone (CTX) following intravenous (IV) and intramuscular (IM) administrations in premature calves. Using a parallel design, 24 premature calves were randomly divided into the two antibiotic groups. Each of the six animals in the first group received CFQ (2 mg/kg) through IV or IM administration. The second group received CTX (20 mg/kg) via the same administration route. Plasma concentrations of the drugs were analyzed by high-performance liquid chromatography and noncompartmental methods. Mean pharmacokinetic parameters of CFQ and CTX following IV administration were as follows: elimination half-life (t1/2λz) 1.85 and 3.31 hr, area under the plasma concentration–time curve (AUC0–∞) 15.74 and 174 hr * μg/ml, volume of distribution at steady-state 0.37 and 0.45 L/kg, and total body clearance 0.13 and 0.12 L hr−1 kg−1, respectively. Mean pharmacokinetic parameters of CFQ and CTX after IM injection were as follows: peak concentration 4.56 and 25.04 μg/ml, time to reach peak concentration 1 and 1.5 hr, t1/2λz 4.74 and 3.62 hr, and AUC0–∞ 22.75 and 147 hr * μg/ml, respectively. The bioavailability of CFQ and CTX after IM injection was 141% and 79%, respectively. IM administration of CFQ (2 mg/kg) and CTX (20 mg/kg) can be recommended at 12-hr interval for treating infections caused by susceptible bacteria, with minimum inhibitory concentration values of ≤0.5 and ≤4 μg/ml, respectively, in premature calves. However, further research is indicated to assess the pharmacokinetic parameters following multiple doses of the drug in premature calves.