The recent progress in ribonucleic acid (RNA) therapy is attracting attention in the fields of personalized nanomedicine and intractable and infectious disease treatments. RNA is unstable and should be delivered to target tissues without RNase degradation, and thus, RNA delivery systems are indispensable in ensuring therapeutic effects. Lipid-based nanoparticles, such as lipid nanoparticles (LNPs) and liposomes, are promising carriers in RNA delivery to target organs. In 2020, Moderna and Pfizer-BioNTech COVID-19 vaccines, which encapsulate messenger RNAs (mRNAs) within LNPs, were approved worldwide and could protect against severe COVID-19. Generally, lipid-based nanoparticles, including LNPs and liposomes, are prepared using various methods, including film hydration, ethanol injection, and detergent removal. After particle preparation, the sizes of the nanoparticles are tuned via sonication or extrusion. Recently, microfluidic-based ethanol injection has been widely used in producing size-controlled nanoparticles without complicated size-tuning processes. In this method, aqueous and lipidcontaining ethanol solutions are introduced into a microfluidic device, and the ethanol is then continuously diluted with the aqueous solution within the device. Lipid molecules aggregate upon ethanol dilution, followed by nanoparticle formation, and thus, ethanol is critical in nanoparticle formation in terms of nanoparticle size, structural change, and drug loading. However, the effects of time-dependent changes in ethanol concentration on lipid-based nanoparticles remain unclear owing to the lack of in situ measurement methods.