The present study focused on those fractions of pine bark extract that were enriched in tri-and tetrameric PACs via a two-steps centrifugal partition chromatography (CPC) based on dentin bioassay guidance. In view of practical limitations posed by bioassay throughput and costs, one valuable means of targeting the isolation of these PACs is to distinguish them from other compounds by following their bright pink-red colored bands on Si gel TLC plates after spraying with vanillin sulfuric acid and heating. A multi-step approach for the further purification of enriched bioactive fractions was applied to isolate the 13 PACs. The approach included Sephadex LH-20 and RP-18 column chromatography with eluents of different selectivity, as well as semipreparative HPLC (see Experimental Section for details).