Determination of vitamin B₆ (pyridoxine hydrochloride) was described using high performance liquid chromatographic method. The analysis was achieved by cosmos IL 5C₁₈-MS-II column (250 mm x 4.6 mm i. d., 5µm particle size) at room temperature. The mobile phase used was Acetonitrile, buffer solution (Citric acid, Na₂HPO₄ pH4) buffer solution in the ratio (70:30) (V: V). the flow rate was set to 1.25 mL.min^-1 and the retention time 1.82 min with UV-detection at 282 nm. Beer's law was obeyed over the concentration range 10-1250 µg.mL^-1. The method was accurate (relative error % less than 0.05%), precise (RSD better than ±1.05%), average recovery 100.05%, with a limit of detection and quantification of 2.2μg.ml^−1, and 7.34μg.mL^−1 respectively. The proposed method was successfully applied to determine the pyridoxine hydrochloride in pharmaceutical preparations in both forms of tablet and injection