AbstractA method for the analytical and semi‐preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi‐synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed‐phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV–vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments. Copyright © 2006 John Wiley & Sons, Ltd.