<jats:title>ABSTRACT</jats:title><jats:p>A high performance liquid chromatographic process was developed for betacyanin and betaxanthin pigments in a fermented red beet root extract. The method employs a reverse‐phase microparticular C<jats:sub>18</jats:sub> column, ion pairing, and a methanol‐water mobile phase, using a solvent gradient program. Spectrophotometric evidence and an enzymatic method were used to unequivocably identify peaks obtained from LC chromatograms. This accurate method for betalains separation and quantification was rapid and efficient. This method of assay enables the stability of the colorant in drug forms to be monitored routinely.