The identification and isolation of bioactive peptides from food sources is a very active research area considering their potential use in both functional food products and pharmaceuticals. In silico tools can be used to predict possible bioactivity of peptides and can aid experimental procedures by narrowing down high number of enzyme combinations for enzymatic hydrolysis of proteins. In this study, tomato seed, which is a valuable industrial waste, was investigated as a potential bioactive peptide source. Nine tomato (Solanum lycopersicum) seed proteins were chosen for in silico analysis to assess their potential ACE inhibitory, DPP-IV inhibitory and antioxidative peptides. After homology assessment, the profiles of potential biological activity of proteins and the simulated enzymatic hydrolysis with fifteen individual enzymes and two enzyme combinations were performed and the released peptides were evaluated. This study demonstrates that tomato seed proteins represent a remarkable source for the generation of bioactive peptides. According to the in silico results, it is predicted that enzymatic hydrolysis with pepsin and pepsin + trypsin combination may produce peptide fragments with high bioactivity. However, it should be noted that the resulting peptides and their activities were predicted under in silico conditions that might be different from experimental conditions.