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This study evaluates the in vitro protein hydrolysis of four microalgae (Tisochrysis lutea, Nannochloropsis gaditana, Tetraselmis suecica and Scenedesmus almeriensis) by intestinal proteases of gilthead sea bream (Sparus aurata) and Senegalese sole (Solea senegalensis). The hydrolysis of protein was monitored at different sampling times by electrophoretic techniques, and the quantification of the free amino acids released by proteases. Overall, S. aurata or S. senegalensis proteases hydrolysed microalgae protein in a similar way. The highest hydrolysis values (coefficient of protein degradation, CPD > 70%) were obtained for Tisochrysis and Nannochloropsis biomasses, which showed a progressive and almost complete proteolysis at the end of the in vitro assay. Tetraselmis and Scenedesmus protein was also hydrolysed, but SDS-PAGE revealed that two protein fractions remained virtually intact at the end of the in vitro assay. The final amount of free amino acids released in vitro by the fish digestive enzymes ranged from 9 to 25 g 100 g protein−1. A linear relationship between CPD and the amount of free amino acids released was found, a fact that suggests that microalgae protein is hydrolysed efficiently by the digestive proteases of both fish species. The present study provides information about the protein availability from selected microalgae, which will aid in the initial evaluation of the microalgae as potential protein sources in feeds of two important farmed fish species.