Conditions were determined for the separation of a complex set of anthocyanins (free aglycones, mono- and multiglycosides and esterified forms) by HPLC. The optimised gradient elution method was then used to carry out qualitative and quantitative analysis of anthocyanin compounds present in the callus tissue ofRudbeckia hirta L. and the tubular flowers of the soil-based plant. The summary content of anthocyanin pigments and the content of the main pigment was identified in the analysed biomass. The method developed is useful for the purposes of monitoring the process of biosynthesis of anthocyanins in tissues obtained through in vitro cultures. The advantages of the method for anthocyanins and its application to other anthocyanin-rich materials are also discussed.