Vesicle formation in a series of amphiphilic sequence-defined polypeptoid block co-polymers comprising a phosphonated hydrophilic block and an amorphous hydrophobic block, poly-N- (2- ethyl)hexylglycine-block-poly-N-phosphonomethylglycine (pNeh-b-pNpm), is studied. The hydrophobic/hydrophilic block ratio was varied keeping the total chain length of the co-polymers constant. A new approach for characterizing the vesicle membrane morphology based on low-dose cryogenic electron microscopy (cryo-EM) is described. The individual low-dose micrographs cannot be interpreted directly due to low signal-to-noise ratio. Sorting and averaging techniques, developed in the context of protein structure determination, were thus applied to vesicle micrographs. Molecular dynamic simulations of the vesicles were used to establish the relationship between membrane morphology and averaged cryo-EM images. This approach enables resolution of the local thickness of the hydrophobic membrane core at the 1 nm length scale. The thickness of the hydrophobic core of the pNeh-b-pNpm membranes increases linearly with the length of the hydrophobic block.