Introduction: The objective of this study was to describe a pharmacokinetic-pharmacodynamic (PK/PD) approach for determination of a rational dosage of ampicillin (AMP) and depletion of the antibiotic residues in milk after intramammary administration to cows. Material and Methods: The cows came from different farms from the Lublin Province area. They (n = 9) received 5 g of the drug, containing 75 mg of AMP sodium in physiological solution, through a syringe tube by intramammary administration. Following single intramammary administration, the milk samples (5 mL) were collected after 2, 4,6,8,10,24, 36, 48, and 60 h. The liquid chromatography-mass spectrometry analysis was performed on the Agilent 1200 system connected to an AB Sciex API 4000™ mass spectrometer. The pharmacokinetic analysis of the concentrations of the antibiotic in milk was performed using software Phoenix ® WinNonlin ® 6.4. Calculations were made in non-compartmental (slopes, highest, amounts, and moments) and compartmental analysis. Results: The pharmacokinetic characteristics of AMP after intramammary administration indicate rapid elimination of the drug from milk. The mean residence time had a several-fold lower value than the designated elimination half-life and amounts to only 3.4 h. The concentration of the drug in the milk dropped relatively quickly and the process was very dynamic. Conclusion: The conducted research confirms the rationale of using the PK/PD model in order to verify the dosing regimen for other antibiotic groups and various indicators of the applied PK/PD model.