Reliable prediction of the 3D structure of self-assembled amphiphilic copolymers is essential for applications in which specificity has to be carefully controlled, as for example in nanomedicine. Since supramolecular assemblies are strongly affected by the chemical nature of block copolymers and the preparation methods, it is essential to understand the influence of such parameters on the self-assembly process. We have now successfully synthesized a library of amphiphilic block copolymers, poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA), and investigated the molecular parameters and self-assembly conditions that generate a specific architecture in particular polymersome. We found that 3D assemblies are strongly affected by the preparation method, but not by the initial concentration of block copolymer and solution pH. The phase diagrams of self-assembly behavior show a strong influence of the hydrophilic to hydrophobic ratio (f(PMOXA)), and the molecular mass of each block. In particular, the formation of polymersomes was possible only for block copolymers with high molecular mass PDMS and low f(PMOXA) values. A combination of very low molecular mass PDMS and small f(PMOXA) values induced formation of worm-like micelles, while low molecular mass PDMS and high f(PMOXA) values generated a mixture of small micelles and spherical particles. The polymersome membranes were characterized by electron paramagnetic resonance, which indicated a multilayer structure (PMOXA outer layer, PDMS middle part, and PMOXA inner layer) with low flexibility and permeability. Addition of detergent increased both the flexibility and permeability of the PDMS block, which was proven to act as a barrier layer for the membranes.