Capsazepine is a competitive capsaicin receptor antagonist. Assay method of capsazepine in biological fluid has not been reported yet. We developed a high performance liquid chromatographic (HPLC) method for the determination of capsazepine in rat plasma. A deproteinated serum sample was subjected to an organic phase extraction procedure. The residue was reconstituted in acetonitrile and then an aliquot was directly injected onto an octadecylsilica column. The mobile phase employed was acetonitrile-water (60% acetonitrile in water, v/v). The flow rate was 1.2 mL/min, and capsazepine elution from the HPLC column was monitored by UV absorption at 234 nm. The retention time of capsazepine and YH439 (internal standard) were 4.6 min and 10.8 min, respectively. The detection limit in rat plasma was 0.05 μg/mL. The mean percentage recovery of the drug in the concentration range of 0.05-5 μg/mL was 97.05%, while the inter-day coefficient of variation of the same concentration range was less than 10%. The method for quantitation of capsazepine in rat plasma was accurate and sensitive for in vivo studies.