1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045) is a new biguanide antimicrobial agent currently in clinical use as a topical bactericidal antiseptic. A method combining high-performance liquid chromatography (HPLC) with electrospray ionization (triple and quadruple stage) tandem mass spectrometry (ESI-MS/MS) was developed to quantify OPB-2045 in human serum. Solid phase extraction was performed on 0.2 ml of sample to ensure a high level of sensitivity before HPLC-ESI-MS/MS analysis. The limit of quantitation for the method was set at 0.05 ng/ml. Intra-assay and interassay precision were less than 13.7%, with a deviation from the expected value of no greater than 10.5% at a concentration range of 0.05 ng/ml to 5 ng/ml. Decomposition of OPB-2045 in human serum did not occur after storage for 15 months at -20 degrees C, even after three repetitions of freezing and thawing. Application of this method was demonstrated in a pharmacokinetic study of OPB-2045 in healthy patient subjects after a single topical application of 5 g/L preparation of its liquid formulation.