Lycopene is a carotenoid found in tomatoes with potent antioxidant activity. The aim of the study was to obtain an extract containing lycopene from four types of tomatoes, validate a quantification method for the extracts by HPLC, and assess its antioxidant activity. Results revealed that the tomatoes analyzed contained lycopene and antioxidant activity. Salad tomato presented the highest concentration of this carotenoid and antioxidant activity. The quantification method exhibited linearity with a correlation coefficient of 0.9992. Tests for the assessment of precision, accuracy, and robustness achieved coefficients with variation of less than 5%. The LOD and LOQ were 0.0012 and 0.0039 μg/mL, respectively. Salad tomato can be used as a source of lycopene for the development of topical formulations, and based on performed tests, the chosen method for the identification and quantification of lycopene was considered to be linear, precise, exact, selective, and robust.