Abstract: A high performance liquid chromatographic assay was developed for determinationof the plasma concentration of hexamethylene bisacetamide(HMBA) in rabbi.Plasma samples wetevortex-mixed with 0.3 ml of methanol and centrifuged at 10000r/min for 5 min. A 20 μl aliquot ofthe supermatant was injected into the HPLC system. Amalysis was performed on a Du Pont Zorbax C18column(250mm×4.6mm,10μm)with a preculumn(50mm×4.6mm,10μm, YWG-C18)。The eluent was methanol—water(30:70),with flowrate of 1.0ml· min^-1 and UV detection at210 nm. The limit of detection was.0μg·mL^-1 in plasma and the calibration curve was linear overthe range 5～1000μg·mL^-1(r=0. 9988).The HPLC assay was applied for studying the pharma-cokinetics of HMBA in rabbit.