This dataset presents the biochemical and functional characterization of protein hydrolysates from the microalgae <i>Chlorella vulgaris</i> (CVB) and <i>Scenedesmus obliquus</i> (SOB), with the primary proposal of establishing these feedstocks as sustainable alternatives to fetal bovine serum in cell culture systems. The nature of the data is experimental and quantitative, ranging from enzymatic hydrolysis parameters using Alcalase® 2.4 L, to detailed molecular characterization via size-exclusion chromatography and proteomic analysis, which identified bioactive peptide sequences such as LPGWL. Furthermore, the dataset encompasses bioactivity assays, featuring angiotensin-converting enzyme (ACE) inhibition metrics, and cytocompatibility tests on the C2C12 cell line, providing metabolic activity data under reduced serum supplementation (1–2.5%). The scope of the dataset thus covers the entire biotechnological chain: from raw biomass to the validation of non-cytotoxic hydrolysates, offering empirical support for the development of culture media with reduced reliance on animal-derived components, with direct applications in the biotechnology and cultured meat sectors. Data restricted due to intellectual property rights. Please contact the authors for further information.