Dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybiphenyl-2,2′-dicarboxylate (DDB) has been shown to improve liver function in chronic hepatitis patients. Despite the fact that the oral bioavailability for DDB appears to be low and variable, the intestinal absorption of the drug is not well understood because of the lack of sensitivity and inadequate separation of DDB from endogenous peaks derived from the serum in previously developed HPLC assays. The present study describes a reliable HPLC method for DDB in serum samples for normal dose human bioavailability trials. A deproteinated serum sample was subjected to a solid-phase extraction procedure. The residue of the Sep-Pak eluent was reconstituted in acetonitrile and an aliquot was directly injected onto an octadecyl silica column (4 μm, 250 x 4.5mm I.D.). The mobile phase consisting of acetonitrile and water (52.5% acetonitrile in water, v/v), was delivered at a flow rate of lml/min, and DDB elution from the HPLC column was monitored by UV absorption at 278nm. The assay was linear in the range of 5-100ng DDB/ml serum with inter-day and intra-day variation less than 14.3 and 13.2 %, respectively. To determine whether the HPLC assay can be utilized in normal dose bioavailability studies, human serum samples (1ml each) were obtained from a typical normal dose bioavailability study (oral capsule for DDB; 15 mg as DDB) and analyzed for DDB. The drug was readily detectable in all samples from 30min to 720min after administration. Therefore, these data indicate that this HPLC assay is readily applicable to a normal dose pharmacokinetic study of DDB in human subjects.