The development of new separation techniques is an important task in protein science. Herein, we describe how anionic and cationic lipid-based liquid crystalline nanoparticles can be used for protein separation. The potential of the suggested separation methods is demonstrated on green fluorescent protein (GFP) samples for future use on more complex samples. Three different CEC-LIF approaches for protein separation are described. (i) GFP and GFP N212Y, which are equally charged, were separated with high resolution by using anionic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (ii) High efficiency (800 000 plates/m) and peak capacity were demonstrated separating GFP samples from Escherichia coli with cationic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (iii) Three single amino-acid-substituted GFP variants were separated with high resolution using an approach based on a physical attached double-layer coating of cationic and anionic nanoparticles combined with anionic lipid nanoparticles suspended in the electrolyte. The soft and porous lipid-based nanoparticles were synthesized by a one-step procedure based on the self-assembly of lipids, and were biocompatible with a large surface-to-volume ratio. The methodology is still under development and the optimization of the nanoparticle chemistry and separation conditions can further improve the separation system. In contrast to conventional LC, a new interaction phase is introduced for every analysis, which minimizes carry-over and time-consuming column regeneration.