Hesperidin (4′-methoxy3′,5,7-trihydroxy flavanone) is a bioflavonoid molecule with remarkable wound healing properties as well as anticancer, antibacterial, antidiabetic, hepatoprotective, anti-inflammatory, and chemoprevention action. As a result, the current study used a stability-indicated reverse phase-high performance liquid chromatography (RP-HPLC) approach to synthesize and analyze hesperidin nanotransferosome. The important elements of the RP-HPLC method for hesperidin-based nanotransferosome stability highlighted were the retention time of 5.45 minutes, the limit of detection (LOD) of 0.008 μg/ml, the limit of quantification (LOQ) of 0.024 μg/ml, and the % relative standard deviation (RSD) of less than 2%. Hesperidin-loaded nanotransferosome were successfully demonstrated using the current RP-HPLC method, which has been proven to be stable. All vital factors, such as accuracy, linearity, LOD, LOQ, precision, and % RSD, were within the appropriate limits. Hesperidin was successfully quantified and estimated using the specified RP-HPLC method, and hesperidin-based nano transferosome confirmed hesperidin stability. This approach will be vital in evaluating the presence of hesperidin-containing therapeutic formulations.