The growing interest of consumers in healthy food challenges growers to continuously improve the nutritional quality of their crops. In this research, the potential of a more saline growth environment for improved antioxidant concentration in tomato fruit was studied, and an U-HPLC-MS/MS method for the determination of lycopene, β-carotene, and α-tocopherol was optimized. Analytes were thereby separated on a 1.9 μm Hypersil GOLD C(18) column and quantified on a TSQ Vantage triple-quadropole mass spectrometer. The method displayed a short analysis time (6 min), a high specificity, and an excellent repeatability (≤6.39%). Furthermore, it was demonstrated that the electrical conductivity level of the applied nutrient solution did not unambiguously influence antioxidant concentration in tomato fruits. Future research should focus on moderate salt stress only and should aim at reducing natural variation by more closely controlling the growth environment and a more objective determination of the ripening degree.