Biapenem, a new parenteral carbapenem, has been widely used for treating bacterial infections.Existing methods to investigate the concentration of biapemen, either lacked sensitivity/reproducibility or had no internal standard as control.Here an improved reversed-phase high-performance liquid chromatographic (RP-HPLC) method was established in human plasma and urine.After adding p-aminobenzoic acid as the internal standard to plasma or urine, plasma samples were ultra-filtrated and urine samples were diluted directly.Chromatographic separations were carried out on a 4.6 mm ×150 mm column with acetonitrile-0.1mol/L sodium acetate (2∶98, v∶v; pH 4.38 or 4.00) as mobile phase and UV detection at 300nm.The extraction recovery was 91.51% for biapenem at the concentration level of 5 μg/mL for plasma and urine, with linear correlation coefficients greater than 0.998.The intra-day and inter-day relative standard deviations (R.S.D.) for biapenem at low, middle and high levels in human samples were less than 12.51% for plasma and less than 7.05% for urine.