AbstractPorous liquid crystalline lipid‐based nanoparticles are shown here to enable protein analysis in microchip electroseparation by reducing sample adsorption. Additionally, higher stability and reproducibility of the separations were observed. The method was tested by separating green fluorescent protein (GFP) in hot embossed cyclic olefin polymer microchips with integrated fiber grooves for LIF detection. The sample adsorption was indirectly quantified by measuring the height, width and asymmetry of the separation peaks for various concentrations of nanoparticles in the sample and background electrolyte. Without nanoparticles, electropherograms displayed typical signs of extensive adsorption to the channel walls, with low, broad tailing peaks. Higher, narrower more symmetric peaks were generated when 0.5–10% nanoparticles were added, showing a dramatic reduction of sample adsorption. The current through the separation channel decreased with nanoparticle concentration, reducing to half its value when the nanoparticle concentration was increased from 0.5 to 4%. Addition of nanoparticles enabled separations that were otherwise hindered by extensive adsorption, e.g. separation of GFP mutants differing by only one amino acid. It was also observed that increasing the nanoparticle concentration increased the number of impurities that could be resolved in a GFP sample. This indicates that the adsorption is further reduced, and/or that the nanoparticles provide an interacting pseudostationary phase for electrochromatography.