Doxycycline is a tetracycline licensed for veterinary use in some countries, but no clinical breakpoints are available for veterinary pathogens. The objectives of this study were i) to establish breakpoints for doxycycline and ii) to evaluate use of tetracycline as a surrogate to predict doxycycline susceptibility of Staphylococcus pseudintermedius.MICs and inhibition zone diameters were determined according to CLSI standards in 168 canine S. pseudintermedius isolates. Tetracycline resistance genes were detected by PCR, and time-kill curves were determined for representative strains. In vitro pharmacodynamic and target animal pharmacokinetic data were analyzed by Monte Carlo simulation (MCS) for development of MIC interpretive criteria. Optimal zone diameter breakpoints were defined using the standard error-rate bounded method.Both drugs displayed bacteriostatic activity and bimodal MIC distributions. Doxycycline was more active on non-wild-type strains than tetracycline. MCS and target attainment analysis indicated a certainty of ≥ 90% for attaining an AUC/MIC ratio of > 25 after standard dosage of doxycycline (5 mg/kg q12h) for strains with MIC ≤ 0.125 μg/ml. Tetracycline predicted doxycycline susceptibility but current tetracycline breakpoints were inappropriate for interpretation of doxycycline susceptibility testing. Accordingly, dog-specific doxycycline MIC (S ≤ 0.125, I = 0.25 and R ≥ 0.5 μg/ml) and zone diameter (S ≥ 25, I= 21-24 and R ≤ 20 mm) breakpoints, and surrogate tetracycline MIC (S ≤ 0.25, I = 0.5 and R ≥ 1) and zone diameter (S ≥ 23, I = 18-22 and R ≤ 17 mm) breakpoints were proposed based on the data generated by this study.