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Among the large variety of messenger RNA (mRNA) delivery systems, those developed with lipid-based formulations were the most widely used and efficient. In our lab, we produced different mRNA formulations made with liposomes, hybrid lipid polymer, and lipid nanoparticles. Our formulations were made with lipids bearing imidazole groups that trigger the endosomal escape of nanoparticles once protonated inside the mild acidic milieu of endosomes upon their cell uptake. Herein, we describe protocols that we used to produce, optimize, and characterize those formulations. The transfection efficiency is influenced by various factors including the physicochemical parameters of the nanoparticles, their efficiency to be internalized in cells, and their intracellular routing as well as their capacity to induce immune system sensors. We provide details on how to quantify the amount of mRNA nanoparticles uptake by cells and evaluate the acidity of the intracellular compartments where they are located, to investigate the endosomal escape, and to assess the activation of innate immune sensors as phosphorylation of PKR hampering mRNA translation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)