Carotenoids are molecules that play important roles in both plant development and in the well-being of mammalian organisms. Therefore, various studies have been performed to characterize carotenoids' properties, distribution in nature and their health benefits upon ingestion. Nevertheless, there is a gap regarding a fast detection of them at the plant phase. Within this contribution we report the results obtained regarding the application of surface enhanced Raman spectroscopy (SERS) toward the differentiation of two carotenoid molecules (namely, lycopene and β-carotene) in tomato samples. To this end, an e-beam lithography (EBL) SERS-active substrate and a 488 nm excitation source were employed, and a relevant simulated matrix was prepared (by mixing the two carotenoids in defined percentages) and measured. Next, carotenoids were extracted from tomato plants and measured as well. Finally, a combination of principal component analysis and partial least squares regression (PCA-PLSR) was applied to process the data, and the obtained results were compared with HPLC measurements of the same extracts. A good agreement was obtained between the HPLC and the SERS results for most of the tomato samples.