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A novel isocratic reverse phase high performance liquid chromatography (RP-HPLC) with photo diode array (PDA) detection method for the determination of dobutamine (DBT) in rat plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Homoveratrylamine was used as an intal standard. Methanol was used as the extracting solvent for the preparation of plasma samples. Samples were separated on a Symmetry C18 (250 mm ? 4.6 mm i.d., 5μm) analytical column. Acetonitrile and 15 mM potassium dihydrogen phosphate (pH 5.0 with 0.3%TEA) (20:80, v/v) was used. The column oven temperature was optimized at 35 1C and the flow rate was 0.8 mL/min. The detection wavelength was fixed at 230 nm for entire analysis. The calibration curve was found to be linear over the concentration range of 50-2000 ng/mL (r2 ? 0.9992). The limit of quantification (LOQ) of the method was 50 ng/mL. The % RSD values of accuracy and precision values for intra and inter days were o15%at quality control (QC) concentrations. Recovery, stability and robustness were studied within the acceptable range according to ICH guidelines. The method was efficiently applied to a pharmacokinetic study in healthy Wistar rats.