Flavonoids do have a lot of pharmacologic effects and health benefits. And are a group of polyphenolic compounds. For the effective delivery of flavonoids, to improve the bioavailability they are being formulated in many ways and for this an accurate analysis in a formulation, a suitable analytical method is needed. For the analysis of flavonoids, High-Performance Liquid Chromatography offers an accurate, sensitive technique. Hence in the present work, an attempt is made to develop and validate an economic, rapid and sensitive method for quantitative determination of Hesperidin in solid lipid nanoparticles based on isocratic reversed phase high-performance liquid chromatography. Chromatography was performed on a C-8 reverse-phase analytical column and photo diode array detector, the following conditions were chosen as optimal: mobile phase – Mobile phase A (methanol : acetonitrile – 60:40) 15% v/v: Mobile phase B (purified water acidified with 0.20 % ortho-Phosphoric acid-pH 2.5) 85% v/v, flow rate – 1.0 mL/min and ambient temperature. Linearity was observed in the concentration range 1-25 µg /mL with a correlation coefficient of 0.999 and the limit of detection (LOD) is 0.25µg/mL and limit of quantification (LOQ) is 0.81µg/mL. The proposed method allowed direct determination of Hesperidin with a high degree of accuracy, precision, robustness, specificity in solid lipid nanoparticles in the presence of formulation excipients. Keywords: Hesperidin, Solid Lipid Nanoparticle Formulation, Quantitative Analysis, Isocratic Reverse Phase High-Performance Liquid Chromatography