Vitamin B6 analysis by reverse-phase high performance liquid chromatography and its validation in milk powder prompted this study. The calibration curves for pyridoxal, pyridoxamine and pyridoxine were evaluated and method precision and accuracy assessed. It was found that the working range was adequate for all the analytes. The analytical procedure was verified by testing the enzymatic efficiency, precision and accuracy. In milk powder samples pyridoxine was the only vitamer found in quantities above the considered limit of quantification and the accuracy showed Z-scores lower than 2. Uncertainty estimation based on method validation results was 0.066 mg/100g, for a vitamin B6 content of 0.61 mg/100 g. It was concluded that the method is adequate for vitamin B6 quantification in powder milk.