<jats:title>Abstract</jats:title><jats:p>A pharmacokinetics/pharmacodynamics (PK/PD) approach was used to determine the best empirical dosage regimen of cefazolin (CEZ) after intramuscular (IM) administration of CEZ in horses. Seven horses received a single IM or intravenous (IV) administration of CEZ of 5 mg/kg bodyweight (BW) according to a crossover design. CEZ plasma concentrations were measured using LC–MS/MS. The plasma concentrations in these seven horses and those of six other horses obtained in a previous study with an IV CEZ dose of 10 mg/kg were modelled simultaneously using NonLinear Mixed‐Effect modelling followed by Monte Carlo simulations to establish a rational dosage regimen. A 90% Probability of Target Attainment (PTA) for a PK/PD target of a free plasma concentration exceeding MIC<jats:sub>90</jats:sub> (<jats:italic>f</jats:italic>T > <jats:sub>MIC</jats:sub>) for 40% of the dosing interval was set for selecting an effective dosing regimen. The typical half‐life of absorption and bioavailability after IM administration were 1.25 h and 96.8%, respectively. A CEZ dosage regimen of 5 mg/kg BW q12h IM administration achieved therapeutic concentrations to control both <jats:italic>Streptococcus zooepidemicus</jats:italic> and <jats:italic>Staphylococcus aureus</jats:italic>. For the same dose, the <jats:italic>f</jats:italic>T > <jats:sub>MIC</jats:sub> after IM administration was significantly longer than after IV administration, and the IM route should be favoured by clinicians for its efficiency and convenience.</jats:p>