Two HPLC methods with diode array detection (HPLC-DAD) and electrospray ionization-mass spectrometry (HPLC-ESI/MS), respectively, were developed to investigate the differences of chemical constituents and their metabolism in gastrointestinal tract in vitro between two decoctions of crude and processed Glycyrrhizae radix. Total of eleven constituents (liquiritin apioside, liquiritin, licuraside, isoliquiritin, ononin, glycyrrhizin, liquiritigenin-7,4'-diglucoside, licorice saponin A3, 22β-acetoxylglycyrrhizic acid, licorice saponin G2, and yunganoside E2) were identified in the two decoctions, whereas lower contents of these constituents were usually found in the decoction of processed Glycyrrhizae Radix. [corrected] Furthermore, these constituents were metabolized into their respective aglycons in human intestinal bacteria juice, and the metabolism ratios were all higher in processed Glycyrrhizae Radix [corrected] decoction. No change was found in artificial gastric or intestinal juice. This study revealed that the processing can alter the contents of main constituents in crude G. radix and their metabolism in gastrointestinal tract, in which intestinal bacteria play an important role in the metabolism of licorice constituents.