Protein from fish is known as the precursor of biologically active peptides that exert various health benefits such as antihypertensive, antitumor, and immunomodulatory properties. Hence, this study aimed to perform an extraction of anchovy, LC-MS/MS analysis and in silico evaluation of the major proteins in anchovies as potential precursors of biologically active peptides in addition to determining whether such peptides can be released by selected proteolytic enzymes. Anchovy was subjected to protein extraction followed by total soluble protein concentration determination using Bradford assay. The sample was subjected to in-solution trypsin digestion, which was then analysed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). A bioinformatic approach by PEAKS Studio was used to identify the protein. A total of four anchovy’s proteins which are myosin light chain 1, V(D)J recombination-activating protein, 60 kDa chaperonin and heat shock protein 90AA1 were identified. Then, the identified proteins were subjected to in silico approach using the BIOPEP database. The biological potential of the theoretically released angiotensin-I converting enzyme (ACE) inhibitory peptides and dipeptidyl peptidase (DPP)-IV inhibitory peptides were predicted by determining the frequency of occurrence of fragments with a given activity. 60 kDa chaperonin and heat shock protein 90AA1 predicted the highest number of biological activities for ACE inhibitory peptides (284 and 264 fragments) and DPP-IV inhibitory peptides (395 and 409 fragments). The most promising enzyme for the generation of bioactive peptides from anchovy protein was anticipated to be pepsin (pH > 2), which theoretically released a high number of DPP-IV inhibitory peptides and ACE inhibitory peptides through the action of in silico proteolysis. Overall, this work highlighted that anchovy protein could be a promising precursor of bioactive peptides that have ACE and DPP-IV inhibitory activities for developing functional food or nutraceutical products.