Microalgae are increasingly recognized as sustainable and versatile sources of high-value bioactive compounds, particularly functional proteins and peptides. Among them, Arthrospira platensis and Tetraselmis chuii stand out due to their high protein content, balanced amino acid profiles, and potential for use in food and health applications. This study aimed to investigate the bioactive potential of peptides derived from these two species through a comprehensive approach combining enzymatic hydrolysis, peptidomic analysis, and in silico bioactivity prediction. Proteins were extracted from A. platensis and T. chuii biomass and hydrolyzed using food-grade pepsin under optimized conditions and varying enzyme-to-substrate (E/S) ratios. The resulting protein hydrolysates—A. platensis protein hydrolysate (APPH) and T. chuii protein hydrolysate (TCPH)—were subjected to peptide profiling using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with tandem mass spectrometry (MS/MS). Identified peptides were then analyzed using multiple bioinformatics tools to assess their molecular weight, hydrophobicity, amphipathicity, potential toxicity, and predicted biological activities. A total of 265 unique peptides were identified, with 187 peptides derived from APPH and 78 derived from TCPH. The peptides exhibited favorable physicochemical properties and bioaccessibility features. In silico predictions highlighted significant multifunctional bioactivities, including antihypertensive, antidiabetic, anti-inflammatory, and antimicrobial potentials. None of the APPH peptides showed any predicted cytotoxic or hemolytic effects, whereas only one peptide from TCPH presented a potential cytotoxicity risk. Notably, the most promising peptides originated from highly conserved proteins such as RuBisCO, ATP synthase subunits, and phycobiliproteins. These findings underscore the promise of A. platensis and
T. chuii as valuable sources of novel, safe, and multifunctional peptides suitable for nutraceutical development. The integrated use of peptidomics and computational screening provides an efficient strategy for peptide discovery and prioritization, paving the way for future validation and application in functional foods, dietary supplements, and health-oriented formulations.