Here we report the quantitative in situ characterization of size distribution evolution of polymeric nanoparticles incubated in murine serum, filtered and unfiltered murine blood. We used an analytical optical approach, named Single Particle Extinction and Scattering (SPES), which relies on the measurements of two independent parameters of single particles. SPES is based on a robust self-reference interference optical scheme which allows a rejection of the spurious signals coming from the background caused by the medium. We employed polystyrene nanoparticles as reference system and polydisperse poly(lactic-co-glycolic acid) nanoparticles. Our results demonstrate that SPES can be used for carrying out ex vivo analysis of nanoparticles to evaluate the modifications that NPs undergo in vivo following different routes of entry. Conversely, Dynamic Light Scattering is not able to provide reliable results for these systems due to the presence of the biological components in solution.