Bicalutamide is a non-steroidal anti-androgen drug used for the treatment of androgen-dependent prostate cancer. Hesperetin is a natural bioflavonoid that can be used in combination with bicalutamide to improve efficacy and decrease tolerance. The aim of the present work was to develop and validate a simple, sensitive, rapid reverse phase-high performance liquid chromatographic method for simultaneous estimation of bicalutamide and hesperetin. The validation parameters such as specificity, linearity, precision and accuracy, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2 (R1) guidelines. Chromatographic separation was achieved on Lichrocart(®) CN column (250 × 4 mm, 5 µm, MERCK) with isocratic elution. The retention times and detection wavelength, for hesperetin and bicalutamide were 4.28 min, 288 nm and 5.90 min, 270 nm respectively. The intra-day and inter-day assay precision and accuracy were found to be <2% over linearity of 50-2000 ng/mL with R(2) 0.999. LOD and LOQ, of bicalutamide and hesperetin was 14.70, 44.57 ng/mL and 16.11, 48.84 ng/mL, respectively. The method was successfully applied for encapsulation efficiency and drug release studies from bicalutamide and hesperetin loaded nanoparticles.