There are concerns that effectiveness and consistency of biopharmaceutical formulations, including vaccines, may be compromised by differences in size, concentration and shape of particles in suspension. Thus, a simple method that can help monitor and characterize these features is needed. Here, nanoparticle tracking analysis (NTA) was used to characterize particle concentration and size distribution of a highly-purified rabies vaccine (RABV), produced in Vero cells without raw materials of animal origin (RMAO). The NTA technique was qualified for characterization of RABV particles by assessing the stability profile of vaccine particles over 5-55 °C. Antigenicity of the viral particle was also monitored with the enzyme-linked immunosorbent assay (ELISA) and NTA. RABV particle size diameters were 100-250 nm (mean:150 nm), similar to sizes obtained when labelled with rabies anti-G D1-25 monoclonal antibody, suggesting mainly antigenic virus-like particles, also confirmed by transmission electron microscopy. Thermal stress at 55 °C decreased the concentration of anti-G D1-25-labelled particles from 144 hours, coherent with conformational changes leading to loss of G protein antigenicity without impacting aggregation. Results from RABV antigenicity assessment during the 24 months monitoring of stability showed good correlation between NTA and ELISA. NTA is a suitable approach for the characterization of biopharmaceutical suspensions.