Carbinoxamine maleate is an antihistamine drug with mild sedation effects, which is used to treat seasonal and perennial allergic rhinitis clinically. In order to optimize drug therapy, reduce drug accumulation, lessen the frequency of adverse effects and facilitate clinical research, a high performance liquid chromatography tandem mass spectrometry assay was firstly established and fully validated for the quantitative admeasurements of carbinoxamine. After extraction with ethyl acetate, the chromatographic separation was implemented on a C18 column (Hypersil GOLD, 100 mm × 2.1 mm, 3.0 μm) using gradient elution with water (containing 0.1% formic acid) and methanol at the flow rate of 0.4 mL/min. The analytes were measured under multiple reactions monitoring (MRM) mode with m/z 291.2 → 202.1 for carbinoxamine and m/z 285.0 → 193.2 for diazepam (IS) using electrospray ionization source (ESI) in the positive ion mode. A satisfactory linearity was obtained over the wide extent of 0.1-100.0 ng/mL (r > 0.99). Inter- and intra-day precision and accuracy of the assay were favorably accorded with the currently recognized limits (< 8.9%). The mean extraction recoveries for carbinoxamine ranged from 74.00% to 86.4%. The pharmacokinetic characteristics of carbinoxamine were subsequently evaluated in beagles. Following intragastric administration (0.534 mL/kg), carbinoxamine possessed a large apparent volume of distribution of the central compartment (Vc = 1005.7 ± 945.9 L/kg), oral clearance (Cl = 112.446 ± 53.249 L/h/kg), and a relatively long absorption time (Tmax = 2.38 ± 1.00 h). This analytical method with adequate sensitivity was applicable to pharmacokinetic study and could monitor concentrations of carbinoxamine in beagle plasma. Moreover, the methodology could be used for further bioequivalence determination and addressing metabolism associated with the drug.