Microalgae are unicellular marine organisms that have promoted complex biochemical pathways to survive in greatly competitive marine environments. They could contain significant amounts of high-quality proteins which, because of their structural diversity, contain a range of yet undiscovered novel bioactive peptides. In this work, a peptidomic platform was developed for the separation and identification of bioactive peptides in protein hydrolysates. In this work, a peptidomic platform was developed for the extraction, separation, and identification of bioactive peptides in protein hydrolysates. Indeed, extraction of proteins from recalcitrant tissues is still a challenge due to their strong cell walls and high levels of non-protein interfering compounds. Therefore, seven different protein extraction protocols, based on mechanical and chemical methods, were tested in order to produce high-quality protein extracts. Proteins obtained by means of the best protocol, consisting of milling the recalcitrant tissue with glass beads, were subjected to enzymatic digestion with Alcalase® and subsequently the hydrolysate was purified by two-dimensional semi-preparative reversed phase liquid chromatography. Fractions were assayed for antioxidant and antihypertensive activities and only the most active ones were finally analyzed by RP nanoHPLC-MS/MS. Around 500 peptide sequences were identified in these fractions. The identified peptides were subjected to an in silico analysis by PeptideRanker algorithm in order to assign a score of bioactivity probability. Twenty-five sequenced peptides were found with potential antioxidant and angiotensin-converting-enzyme-inhibitory activities. Four of these peptides, WPRGYFL, GPDRPKFLGPF, WYGPDRPKFL, SDWDRF, were selected for synthesis and in vitro tested for specific bioactivity, exhibiting good values of antioxidant and ACE-inhibitory activity. [Figure not available: see fulltext.].