Unilamellar phospholipid vesicles prepared using the polymerizable lipid bis-sorbylphosphatidylcholine (bis-SorbPC) yield three-dimensional nanoarchitectures that are highly permeable to small molecules. The resulting porous phospholipid nanoshells (PPNs) are potentially useful for a range of biomedical applications including nanosensors and nanodelivery vehicles for cellular assays and manipulations. The uniformity and size distribution of the pores, key properties for sensor design and utilization, have not previously been reported. Fluorophore-assisted carbohydrate electrophoresis (FACE) was utilized to assess the nominal molecular weight cutoff limit (NMCL) of the PPN via analysis of retained dextran with single monomer resolution. The NMCL of PPNs prepared from pure bis-SorbPC was equivalent to a 1800 Da linear dextran, corresponding to a maximum pore diameter of 2.6 nm. Further investigation of PPNs prepared using binary mixtures of bis-SorbPC and dioleoylphosphatidylcholine (DOPC) revealed a similar NMCL when the bis-SorbPC content exceeded 30 mol %, whereas different size-dependent permeation was observed below this composition. Below 30 mol % bis-SorbPC, dextran retention provided insufficient mass resolution (162 Da) to observe porosity on the experimental time scale; however, proton permeability showed a marked enhancement for bis-SorbPC ≥ 10 mol %. Combined, these data suggest that the NMCL for native pores in bis-SorbPC PPNs results from an inherent property within the lipid assembly that can be partially disrupted by dilution of bis-SorbPC below a critical value for domain formation. Additionally, the analytical method described herein should prove useful for the challenging task of elucidating porosity in a range of three-dimensional nanomaterials.