Tomato products and especially concentrated tomato paste are important sources of antioxidants in the Mediterranean diet. Tomato fruit contain well-known antioxidants such as vitamin C, carotenoids, flavonoids, and hydroxycinnamic acids. The industrial processing of this fruit into tomato paste involves several treatments that potentially affect the final profile of antioxidants and other metabolites in the commercial product. Here we have used both biochemical and metabolomic techniques to assess the effect of each separate step in the industrial production chain starting from fresh fruit to the final tomato paste. Material was collected from five independent tomato paste production events spread over two successive years. Samples comprised the intact ripe fruits and semifinished products after fruit-breaking, separation of the pulp from skin and seeds, evaporation, and finally after canning and pasteurization. The effect of each processing step was determined by different types of analysis. First, the total antioxidant capacity and total phenolic content were determined by commonly used spectrophotometric methods. Second, individual antioxidants in the extracts were identified and compared using an HPLC with online antioxidant detection. Third, in each sample the levels of the major individual antioxidants present, i.e., vitamin C, phenolic compounds (such as rutin and chlorogenic acid), tocopherols, and carotenoids, were quantified. Fourth, an untargeted metabolomic approach using LC-QTOF-MS was used to identify those production steps that have the largest impact on the overall metabolic profile in the final paste as compared to the original fruits. This multifaceted approach has revealed that each processing step induces specific alterations in the metabolic profile, as determined by the different analysis procedures, and that in particular the fruit-breaking step and the removal of seed and skin significantly affect the levels of antioxidants and many other metabolites present in commercial tomato paste.