In this study, the authors developed a new HPLC-UV/VIS method for the quantitation of Lycopene in tomatoes. The separation was carried out on a reversed phase C30column (10 mm x 250 mm, 5 um) with the mobile phase of methyl -tert butyl ether: methanol (75:25, v/v), flow rate of 1.5 mL/min, wavelength of 472 nm. The linear range of the method was 0.02-66.67 ug/mL. LOD and LOQ of the methods were 8,0 and 16,0 ng/mL, respectively. The precision and accuracy of the method were 2.35 percent - 4.87 percent and 97.6 percent - 103.4 percent, respectively. The method was the applied to the quantitation of 20 tomatoes samples purchased from markets, the contents of Lycopene in Tomatoes varied from 3 to 12 mg/100 g based on fresh weight.