Here, we introduce a novel method for the analysis of LNPs by AF4-MALLS785nm-DLS-RI that aims to attain authentic particle size distributions and particle concentrations. The separation efficiency of the AF4 system (i.e., peak shape and resolution) was improved by increasing the Vx from 0.3 to 0.7 mL min -1 and downsizing the channel spacer from 350 to 250 µm. Also, reducing the laser power of the MALLS detector to 20% avoided signal clipping (i.e., exceeding of the upper detector range), due to the high scattering intensities of LNPs. The optimized AF4 system was able to accurately determine particle size values from MALLS785nm (i.e., Rg,n, Rg,w, Rg,z Rg,avg).