This study aimed to identify and characterize bioactive peptides derived from protein hydrolysates of Arthrospira platensis (APPH) and Tetraselmis chuii (TCPH) using an integrated peptidomics and bioinformatics approach. Proteins extracted from the microalgae were hydrolyzed using pepsin (EC 3.4.23.1) at various enzyme/substrate (E/S) ratios. APPH and TCPH, prepared at an E/S ratio of 1/10 (w/w), were analyzed using peptidomics through reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with tandem mass spectrometry (MS/MS). Using the UniProtKB database, a total of 265 unique peptides were identified, including 187 peptides from APPH and 78 peptides from TCPH. Subsequent in silico analysis of these peptides revealed favorable physicochemical properties, with a notable distribution of hydrophobic (APPH: 26; TCPH: 5), amphipathic (APPH: 70; TCPH: 16), and hydrophilic peptides (APPH: 59; TCPH: 17). Toxicity assessments confirmed that none of the peptides showed hemolytic or cytotoxic risks, except for one peptide identified in TCPH with potential cytotoxicity. Furthermore, bioactivity predictions demonstrated significant multifunctional properties (scores exceeding the 0.500 threshold), identifying peptides with antihypertensive (APPH: 2; TCPH: 1), anti-diabetic (APPH: 2), anti-inflammatory (APPH: 14; TCPH: 5) and antimicrobial (APPH: 7) activities. The current study thus establishes protein hydrolysates from A. platensis and T. chuii as promising sources of bioactive peptides suitable for nutraceutical applications. Our integrated analytical and computational strategy provides critical insights into peptide multifunctionality, supporting further research and development of microalgae-derived peptides.