A simple, rapid and reliable direct spectrophotometric method for the determination of hesperidin is proposed and validated. The influence of wavelength, solvent, the ionic strength, pH and temperature on hesperidin determination were investigated. Under the optimum conditions, λ = 283 nm, 60% methanol as the solvent, ionic strength, I = 2.5 × 10-5 mol L-1, pH = 6.4 and T = 37.0 °C, the Beer's law is obeyed in the concentration range 1.83-24.5 µg mL-1. The molar absorptivity and Sandells sensitivity were found to be 1.8 × 104 L mol-1 cm-1 and 0.03 µg cm-2, respectively. The sensitivity of the proposed method was 0.9 µg mL-1 (as limit of detection) and 3.2 µg mL-1 (as limit of quantification). Applicability of the proposed method to the direct determination of total flavonoids as hesperidin equivalents in pharmaceutical formulation (Vitamin C with citrus bioflavonoids & Rose Hips) was demonstrated. Although the presence of ascorbic acid may cause problem in identification and measurements, hesperidin has been determined successfully.