Microalgae are widely used as dietary supplements. Despite their recognized nutritional value, it remains unclear whether consumption of microalgal biomass provides sufficient nutrient bioaccessibility to meet human dietary needs. In this work, the microalga Chlorella vulgaris CCAP 211/11B was cultivated and subjected to Pulsed Electric Fields (PEF) pre-treatment (20 kV·cm⁻¹, 100 kJ·kg⁻¹). The effects of PEF pretreatment were evaluated by assessing protein bioaccessibility throughout the different gastrointestinal (GI) digestion phases, as well as the amino acid profile and cytotoxicity in Caco-2 cells. During the oral and gastric phases, no significant differences (p > 0.05) in protein release were observed between PEF-treated and untreated biomass. At the final intestinal stage, however, protein release was 21.7% in PEF-treated biomass compared with 14.7% in untreated samples, corresponding to a 47% increase in protein bioaccessibility due to PEF pretreatment. Intrinsic fluorescence spectroscopy did not clearly discriminate treatments due to the dominant signal from digestive enzymes. Beyond improving protein availability, PEF-treated C. vulgaris exhibited no cytotoxic effects, confirming the safety of this processing approach for food applications aimed at enhancing protein uptake. Simulated GI digestion of both untreated and PEF-treated samples resulted in comparable amino acid profiles, with tyrosine, methionine, leucine, and lysine detected at the highest relative levels. Overall, these findings demonstrate that PEF treatment represents a promising strategy to enhance protein bioaccessibility in Chlorella vulgaris, thereby strengthening its potential as a functional and sustainable food supplement.