Betaxanthins, the yellow-orange water-soluble pigments from yellow beet (Beta vulgaris ssp. vulgaris cv. Bejo Zaden) and cactus pear (Opuntia ficus-indica cv. Gialla) have been investigated using an HPLC system compatible with mass spectrometry. Five novel betaxanthins were found and characterized as the immonium adducts of betalamic acid with serine, gamma-aminobutyric acid, valine, isoleucine, and phenylalanine. To enable concentration of betalain samples, desalting was performed by solid-phase extraction. With this technique, betacyanins could be separated from the betaxanthins using the pH-dependent retention characteristics of red and yellow betalains. The betaxanthin fraction was taken for the preparation of betalamic acid as a precursor for semisynthetic standards. The HPLC method was applied to yellow beet and cactus pear, revealing a more complex betalain profile than described earlier, thus proving its suitability for screening of betaxanthin-containing plants as potential sources for natural food colors.