A sensitive and selective reverse-phase high performance liquid chromatographic method with fluorescence detection has been developed for determination of capsaicin (8-methyl-N-vanillyl-(trans)-6-nonenamid) and dihydrocapsaicin (8-methyl-N-vanillylnonanamide) in samples generated in rat small intestine luminal perfusion experiments. The experiments were designed to study the biotransformation of capsaicinoids in the small intestine in the rat. The chromatographic separation was performed at room temperature on a ZORBAX Eclipse® XDB-C8 column using isocratic elution with a mobile phase consisting 0.05 M orthophosphoric acid solution and acetonitrile (60:40, v/v; pH 3.0) with a flow rate of 1.5 mL/min. Fluorescence detection was performed at excitation and emission wavelengths of 230 and 323 nm, respectively. The method was evaluated for a number of validation characteristics (accuracy, repeatability and intermediate precision, limit of detection, limit of quantification and calibration range). The limit of detection (LOD) was 50 ng/mL and the limit of quantification (LOQ) was 100 ng/mL for both capsaicin and dihydrocapsaicin reference standards dissolved in blank perfusate. The method was successfully applied for investigation of intestinal absorption of capsaicin and dihydrocapsaicin while 30 μg/mL standardized Capsicum extract – containing capsaicin and dihydrocapsaicin – was luminally perfused for a 90 min period. The structure of the glucuronide metabolites of capsaicin and dihydrocapsaicin appeared in the perfusate was identified by mass spectrometry.