A sensitive, efficient and stable bioanalytical method has been developed and validated for determination of brexpiprazole in dog plasma with UPLC-MS-MS for the first time. Brexpiprazole and internal standard were extracted from plasma samples by liquid-liquid extraction and separated on an Acquity UPLC BEH C18 column. A gradient elution program was developed employing methanol and 10 mM ammonium acetate aqueous solution as mobile phases. The method was validated for parameters of selectivity, LLOQ, linearity, accuracy, precision, matrix effects and stability in accordance with the regulatory guidance on bioanalytical method validation. The validated method was applied in evaluating the pharmacokinetic profiles of brexpiprazole in beagle dogs after a single-dose oral administration of a 4 mg tablet.