Assembly and fusion between different populations of lipid nanoparticles was mediated by membrane-anchored lipidated nucleic acid (LiNA) strands and observed using surface plasmon resonance (SPR) as a label-free real-time assay. Irreversible membrane fusion was distinguished from reversible assembly by enzymatical cleavage of dsDNA tethers in situ. The assay enables user-friendly monitoring and application of membrane fusion in the context of liposomal drug delivery or synthetic biology.