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In order to improve the efficacy of penicillin injection during caesarean section, we aimed to identify the optimal timing of its preoperative administration. A study was conducted in 12 adult, non-pregnant Belgian Blue cows. To evaluate the plasma penicillin concentrations, blood samples were taken from the jugular vein at −5, 15, 30, 45, 60, 120, 240, 480 min relative to the intramuscular (IM) injection of 21,000 IU/kg of body weight of penicillin G. Results showed that plasma concentrations at 15 min after IM injection (668.3 ± 73.7 ng/mL) largely exceeded the minimal inhibitory concentration (MIC) of penicillin-sensitive bacteria (MIC < 125 ng/mL). With increasing time, plasma concentrations continued to rise, attaining an increasing proportion of moderately sensitive bacteria (250 ng/mL > MIC < 2000 ng/mL). The maximal concentration was reached between 1 and 4 h (average: 1.495.1 ± 181.7 ng/mL) after IM injection in the majority of cows, and decreased non-significantly to 1002.1 ± 93.2 ng/mL at 8 h. In conclusion, plasma penicillin concentrations at 15 min after an IM injection inhibit penicillin-sensitive bacteria. However, in order to obtain the maximal protective effect of the antibiotherapy, surgery should be started at 1 to 2 h after IM penicillin injection.