High performance anion exchange chromatography and pulsed amperometry were used to separate and quantify peanut sugars extracted with methanol: chloroform:water (60:25:15, V/V/V), a highly polar solvent which solubilizes other seed components including amino acids. Free sugars were separated on an anion exchange column using a sodium hydroxide gradient and detected with a pulsed amperometric detector equipped with a gold electrode. Free amino acids in the seed extract interfered with sugar analysis by causing peak shifting and co-elution of some amino acids with sugars. Free arginine co-eluted with inositol resulting in a peak area which was 42% of the actual total area for both compounds. Proline co-eluted with fructose. Serine eluted on the leading edge of sucrose. Peak areas of these interfering amino acids were either “additive” or “subtractive” to sugar peaks. Altering the gradient elution or using cation syringe filters to remove contaminant amino acids permitted the accurate identification and quantification of peanut seed sugars. Results from this study suggest applications that can be applied in other biological systems containing free amino acids and sugars.