In this present study, a stereospecific HPLC method that could separate two enantiomers (R/S epimer) of naringenin in bottom base has been developed and validated, and then further applied it to determine stereoselective pharmacokinetics of naringenin in rats. In this method, a normal phase column (Chiralpak AD-H) was used and mobile phase consisting of n-hexane, isopropanol, and trifluoroacetic acid with a gradient elution program. The ultraviolet detection wavelength was at 288 nm and injection volume was 20 μL. The column temperature was at 30 °C, and flow rate was 0.8 mL/min. The validation of the method showed that the calibration curves in plasma were linear ranging from 0.05 to 20 μg/mL for each enantiomer with correlation coefficients of 0.9993 and 0.9997, respectively. The inter-day assay and intra-day assay accuracies (%) for both enantiomers were between 100.89-108.33%, while the inter- and intra-day assay precisions (%RSD) were between 1.99-7.71%. Extraction recovery, elution, and stability of both enantiomers in plasma were evaluated too, and the results showed that the method was reliable. The method was applied to determine the pharmacokinetic profile of two enantiomers of naringenin in rats after intravenous and oral administration. Naringenin has a bioactive effect as antioxidant, anti-inflammatory and immune system modulator and study on its pharmacological potency is becoming popular. The variety of naringenin's bioactivity might be due to its feature of chiral structure according to some reports, the availability of a stereospecific analytical method will be of great help to interpret the findings in different areas.