We present a simple and validated HPLC method for measuring Vitamin B6 urinary metabolite 4-pyridoxic acid (4-PA). Urine samples were treated with 6% perchloric acid. HPLC separation was achieved using Waters column (Symmetry® C18 250 mm × 4.6 mm i.d (5 μM). The mobile phase was a mixture of methanol and 35 mM sodium phosphate buffer containing 2.5 mM sodium heptane polysulfonate at pH=3.5 with 85% O-phosphoric acid. Retention times were 8.0 min for 4-pyridoxic acid. Detection (UV at 302 nm) of analyte was linear in a range from 0.0125 to 0.8 μM. This method is stable, reproducible and has practical advantages such as ease and low cost.