Abstract: A rapid, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of clevidipine butyrate and its primary metabolite clevidipine acid in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm × 4.6 mm, 5 µm) with a gradient mobile phase consisting of methanol and 5 mmol·L^-1 ammonium formate. A chromatographic total run time of 13.0 min was achieved. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 M-H^-→ m/z 234.1 for clevidipine butyrate, m/z 354.0 M-H^-→ m/z 208.0 for clevidipine acid and m/z 256.1 M-H^-→ m/z 227.1 for elofesalamide (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The linear calibration curves for clevidipine butyrate and clevidipine acid were obtained in the concentration ranges of 0.5-100 ng·mL^-1 and 1-200 ng·mL^-1, separately. The lower limit of quantification of clevidipine butyrate and clevidipine acid were 0.5 ng·mL^-1 and 1 ng·mL^-1. The intra and inter- assay precisions were all below 12.9%, the accuracies were all in standard ranges. Stability testing indicated that clevidipine butyrate and clevidipine acid in dog blood with the addition of denaturant methanol was stable under various processing and/or handling conditions. The validated method has been successfully applied to a pharmacokinetic study of clevidipine butyrate injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg·h^-1 for 0.5 h.