Biapenem, a new parenteral carbapenem, has been widely used for treating bacterial infections. A simple, effective and accurate method based on solid-phase extraction (SPE) and HPLC was developed for the quantitative determination of biapenem in human plasma. Stability and feasibility of the method was validated through a series of experiments. Using Vitamin B6 as an internal standard, analyte was separated on a Capcell Pak C18 column after SPE on Oasis hydrophilic-lipophilic balance (HLB) cartridge. The mobile phase was comprised of 0.05 mol/L NaH2PO4 (pH 5.7) and methanol (98:2, v/v) at a flow rate of 1.0 mL/min. Ultraviolet absorbance was measured at 300 nm. The calibration curve was linear in the concentration range of 0.04-50.00 μg/mL, and the lower limit of quantification was as low as 0.04 μg/mL. Recovery rates of biapenem at 0.10, 5.00, and 25.00 μg/mL were about 70%. The validated method has been successfully applied for quantifying biapenem in human samples and a pharmacokinetic study of 12 healthy volunteers who received three different doses (150, 300 and 600 mg) of biapenem by intravenous infusion. Our method has featured good accuracy and precision, and the processed sample was stable. Therefore, it can be propagated for clinical use.