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Background: Trans-resveratrol is a naturally occurring polyphenolic compound extensively marketed as a nutraceutical. It is highly explored molecule in the research community with vast therapeutic potential. Therefore, many inventions were made by pharma-industries for its clinical use and to bring it to the market as a drug. Objectives: In this study, a simple, rapid and sensitive Reverse Phase High Performance Liquid chromatographic (RP-HPLC) method for the quantitative determination of resveratrol was optimized and validated. Materials and Methods: HPLC system of Shimadzu Corporation, Japan, SPD-M201 model connected to Photo-diode Array was used in this study. Intek chromosol C18 column was used to perform the separation. The developed method was validated as per International Conference on Harmonization guidelines. The developed method is further used to estimate the % entrapment efficiency in the bone-targeted resveratrol loaded nanoparticles. Results: The optimum chromatographic separation was achieved by the mobile phase consist of acetonitrile and 25 mM ammonium acetate buffer (pH 4.5) in 45:55 ratios respectively. The flow rate of 0.9 mL/min with a standard retention time of 5.8 min at a wavelength 307 nm was optimized. The RP-HPLC method was developed, optimized and validated with the help of Design of Experiment software using 2³ full factorial design. The desirability value was near to 1, specifies that the method is the ideal and robust. The method is further used to determine the % entrapment efficiency in the bone-targeted resveratrol loaded nanoparticles and found to be 79.27±5.54%. Conclusion: The present analytical method can be used for the quantification of resveratrol in pharmaceutical preparations and dietary supplements. [ABSTRACT FROM AUTHOR]