A highly selective, sensitive and rapid hydrophilic interaction liquid chromatographic method was developed and validated for determination of vesnarinone on dried blood spots. Naftopidil was used as an internal standard. The chromatographic separation was achieved on a reversed-phase zwitterionic hydrophilic interaction liquid chromatographic ZICWHILIC-C18 (4.6 × 100 mm; 5 mm) column using acetonitrile: 10 mM potassium dihydrogen phosphate (pH 4.0; 70 : 30, v/v) as a mobile phase in an isocratic elution mode at a flow rate 0.8 mL min⁻¹ at 27 °C. Photodiode array detection wavelength was set at 247 nm to monitor the column eluents. The method was validated for accuracy, precision, linearity and selectivity by design of experiments following ICH guidelines. The assay exhibited a linear range of 30–2000 ng mL⁻¹ for vesnarinone on dried blood spots. The lower limit of detection was found to be 30 ng mL⁻¹. The intra- and inter-assay coefficients of variation did not exceed 11.46% deviation of the nominal concentration. The recovery of vesnarinone from dried blood spots was >95.0% and its stability were excellent with no evidence of degradation during sample processing for at least 3 months storage in a freezer at −20 °C. The method was successfully applied to a pharmacokinetic study of vesnarinone in rats.