Fish protein hydrolysates are an important source of antioxidant peptides. Electrically driven membrane fractionation called electrodialysis with filtration membrane (EDFM) is a separation technology based on molecular charge and mass, which can fractionate active peptides from complex hydrolysates. This work aimed to evaluate the feasibility of sequential EDFM process for separation of cationic (CP) and anionic (AP) peptides from rainbow trout frame protein hydrolysate, and determine their antioxidant properties. The concentrations of CP and AP increased in the recovery solution, reaching 156 and 85 μg/mL, respectively, after 4-hour treatment, with migration rates of 19.55 ± 2.19 and 10.94 ± 0.39 g/m2h. The CP separation was approximately 50% energy efficient than AP. Both CP and AP fractions were enriched with peptides with DPPH and ABTS radical scavenging properties. The results showed that two-step EDFM process is feasible for recovery and concentration of antioxidant peptides from rainbow trout protein hydrolysate. Industrial relevance The electro-membrane fractionation developed in this study is a two-step process, which is able to selectively separate antioxidant peptides from enzymatic protein hydrolysates based on charge and size. With this particular raw material as the feed, we have shown that using this approach lead to the highest peptide migration rate and a significant improvement in antioxidant activities of both peptide fractions. In addition, this technique is very selective, and environmental friendly as it requires no use of solvent and consumes less energy compared to conventional chromatographic techniques, and thus can be used as a green technology for the fractionation of bioactive peptides from a complex mixture of protein hydrolysates.