The current work was outlined to decide kinetic profile and bioavailability of ceftiofur sodium at measurements of 2.2 mg/kg. b.wt. after a single intravenous and subcutaneous injection in pre-weaned calves. Five clinically typical pre-weaned calves were utilized in this work. Serum ceftiofur concentrations were decided by utilizing high performance liquid chromatography (HPLC) strategy. The serum concentration-time curve has shown a two compartment open model. Taking after a single intravenous injection, distribution half-life (t_0.5α) was 0.11 ± 0.01h, volume of distribution (V_dss) was 0.16 ± 0.004 L/kg, elimination half-life (t_0.5β) was 11.07 ± 0.12h and total body clearance (CL_tot) was 0.013 ± 0.005 L/kg/h. Taking after a single subcutaneous organization, ceftiofur  had a peak serum concentration (C_max) 6.64 ± 0.08 μg/ml at a time (t_max) of 3.38 ± 0.069 h, disposal half-life (t_0.5el) was 2.11 ± 0.25h demonstrating the propensity of calves to eliminate ceftiofur in slow rate. Bioavailability was 77.04 ± 1.64% after subcutaneous injection indicating a good absorption of ceftiofur. Ceftiofur serum concentrations along 36 h following subcutaneous injection in the current work were exceeding the MICs of different susceptible microorganisms responsible for serious disease problems. These results demonstrating successful use of ceftiofur in pre-weaned calves.