A method to determine inositol content in milk using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was developed and validated. Samples were hydrolyzed with 10 mL HCl in a vacuum oven at 120oC, prolonging 6 h. Hydrolyzed samples were neutralized to a pH from 6 to 7.5 before analysing in an HPAEC-PAD system. The analytical program used Dionex CarboPacTM MA1 column (4 × 250 mm) with a mobile phase of a gradient program including NaOH 50 mM and NaOH 1M. The method was validated following AOAC guidelines: selectivity, linear range from 0,01 to 20 mg/L with a coefficient (R) 0.9998, the recoveries in the range of 99 - 102%, and repeatability with RSD under 1.8%. The limit of detection (LOD) and the limit of quantification (LOQ) of inositol were 0.047 µg/g and 0.155 µg/g, respectively. The method has been applied to determine of inositol content in milk samples with content ranging from 22.5 mg/100g to 64.7 mg/100g.