A simple and reliable high-performance liquid chromatographic (HPLC) method for analyzing chlorhexidine in human serum was developed. After the addition of an internal standard, levomepromazine, 0.2 mL serum was deproteinized with 10% perchloric acid. The acidic supernatant was neutralized with 1M potassium carbonate solution, and the insoluble salt was removed by centrifugation. An aliquot of the supernatant was applied to HPLC with UV detection (260 nm). HPLC separation was achieved on a polymer-coated ODS column equilibrated with acetonitrile/water containing 0.05% trifluoroacetic acid, 0.05% heptafluorobutyric acid, and 0.1% triethylamine (40:60, v/v). The calibration curve was linear in the concentration range from 0.05 to 50.0 µg/mL, and the lower limit of detection was 0.05 µg/mL. The accuracy and precision of the method were evaluated at concentrations of 0.5 µg/mL and 5.0 µg/mL. The coefficients of variation ranged from 4.0 to 4.5%. The concentration of chlorhexidine in the serum of a patient who died after a suspected intravenous injection of chlorhexidine gluconate was determined.