The purpose of this paper is to optimize an HPLC method for the determination of lycopene and β-carotene in vegetables and compare it with a spectrophotometric standard method. Among the different conditions studied the most suitable ones for our samples were: extraction with hexane/acetone/ethanol (50:25:25 v/v/v), evaporation of the hexane layer, dissolution of the dry extract in THF/ACN/methanol (15:30:55 v/v/v) and injection on a C18 column with methanol/ACN (90:10 v/v)+TEA 9μM as mobile phase (Φ=0.9ml/min) and λ_detection=475nm. Samples considered for analysis were: tomato, carrot, pepper, watermelon, persimmon and medlar. The HPLC method proposed showed adequate reproducibility (RSD<10.5%), accuracy (100–109% recovery) and sensitive detection limits (0.6μM for lycopene; 0.3μM for β-carotene), with a simple preparation of the samples (one step direct extraction) and short run times (10min) for the quantification of lycopene and β-carotene.