Membrane-delimited compartments, as lipid vesicles, are ubiquitous in natural and synthetic systems. The mechanical properties of such vesicles are crucial for several physical, chemical, and biological processes. However, their accurate determination is still challenging and requires sophisticated instruments and data analysis. Here we report the first evidence that the surface plasmon resonance (SPR) of citrated gold nanoparticles (AuNPs) adsorbed on synthetic vesicles is finely sensitive to the vesicles’ mechanical properties. We leverage this finding to demonstrate that the spectrophotometric tracking of the SPR provides quantitative access to the stiffness of vesicles of synthetic and natural origin, such as extracellular vesicles (EVs). This plasmon-based “stiffness nanoruler” paves the way for developing a facile, cost-effective, and high-throughput method to assay the mechanical properties of vesicles of nanometric size and unknown composition.