Lycopene is highly unsaturated, open-chain carotenoid found abundantly in tomatoes. Research interests into properties & health benefits of lycopene have been growing, bringing attention to content of lycopene in food. The purpose of this work was to develop efficient & accurate protocol that can measure lycopene using HPLC, and establish relationship with Vis/NIR reflectance spectroscopy measurement. Accordingly, mobile phase composition ACN/MeOH (50:50, v/v)+ TEA 9 æM; extraction solvents hexane/acetone/ethanol (50:25:25, v/v/v); re-dissolving residue in THF, followed ACN/MeOH (15:30:55, v/v/v); flow rate 0.6 mL/min and ?detection 472 nm were showed most suitable for lycopene determination in tomato. This method has achieved best characteristic spectra profile with precision (RSD > 15), accuracy & recovery (? 81.7%) and sensitive detection limit (0.0156 ?g/mL) within separation of ~ 21 minutes. Finally, the established method was applied to selected tomato cultivars and a wide range of lycopene content 1.91 to 8.23 mg/100g was observed. Besides, although HPLC is most accepted method; Vis/NIR combined with multivariate techniques has showed potential for determining lycopene.