Ropivacaine is a local anesthetic commonly used in veterinary anesthesia. A liquid chromatography-mass spectrometry (LC-MS) method was developed to quantify free and total ropivacaine in dog plasma, which included rapid equilibrium dialysis. The method was validated for selectivity, specificity, matrix effect, calibration curve and range, accuracy and precision, carry-over, stability, and reinjection reproducibility according to the International Conference on Harmonization M10 guidelines. After ultra-high performance liquid chromatographic (UHPLC) separation, detection and quantification of ropivacaine was performed using a triple quadrupole tandem mass spectrometer with electrospray ionization. LC-MS method validation was carried out in a range of 0.05-1000 ng/mL ropivacaine in dog plasma in two dilutions (1:1 and 1:4). The precision and accuracy of the method were determined at four concentration levels and ranged from 0.40% to 5.30% and 85.50% to 113.30%, respectively. The lower limit of quantification was as low as 0.30 and 0.05 ng/mL, for the quantitation of protein-bound (1:4) and free (1:1) ropivacaine, respectively. All validation parameters met acceptance criteria. This UHPLC-MS/MS method was successfully applied in a clinical study that involved the intraperitoneal instillation of ropivacaine to anesthetized dogs and can be used to quantify free and total ropivacaine in dog plasma.