Red blood cells (RBC) can act as carriers for therapeutic agents and given their biocompatibility and long lifespan in the circulation, they can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBC integrity and lifespan is important for the efficacy of RBC as drug carrier and can be a complex challenge of drug encapsulation. We investigated the impact of drug encapsulation by hypotonic dialysis on RBC physiology and integrity. Several parameters were compared between processed RBC loaded with L-asparaginase (“eryaspase�), processed RBC without drug and non-processed RBC. Processed RBC were less hydrated and displayed a reduction of intracellular content, relative to non-processed RBC. This reduction of intracellular content changed the RBC metabolomic profile, as indicated by the activation of the pentose phosphate pathway, but no impact on the global RBC proteomic profile was observed. We found that the encapsulation process caused moderate morphological changes and was accompanied by an increase of microparticles. Despite a decrease in their deformability, processed RBC were not mechanically retained in a spleen-mimicking device. Moreover, processed RBC had increased surface-to-volume ratio and osmotic resistance. Finally, the half-life of processed RBC was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBC prolonged its in vivo half-life compared to free form. Overall, our study demonstrated that encapsulation by hypotonic dialysis may affect certain  several characteristics of RBC, but does not significantly impact on the in vivo longevity of RBC or their drug carrier function.