Polymers based on 2-oxazoline, such as poly(2-ethyl-2-oxazolines) (PETOx), are considered to be a type of 'pseudopeptide' with the ability to form novel biomaterials. The hydrolysis of PETOx was carried out to evaluate its use in biomedical applications. In the present work, PETOx samples with a range of molar masses were prepared by living cationic polymerization. Hydrolysis was carried out at time intervals ranging from 15 to 180 min to prepare copolymers with different amounts of ethylene imine units. (1)H NMR spectroscopy was used to identify the structure of the hydrolyzed polymers. The dependence of in vitro cell viability on the degree of hydrolysis was determined using three different model cell lines, namely, mouse embryonic 3T3 fibroblasts, pancreatic βTC3 cells, and mouse lymphoid macrophages P388.D1. It was demonstrated that increasing the degree of hydrolysis decreased cell viability for all cell types. Fibroblast cells displayed the highest tolerance; additionally, the effect of polymer size showed no observable significance. Macrophage cells, immune system representatives, displayed the highest sensitivity to contact with hydrolyzed PETOx. The effect of polymer hydrolysis, polymer concentration and the incubation time on cell viability was experimentally observed. Confocal laser-scanning microscopy provided evidence of cellular uptake of pyrene-labeled (co)polymers.