While extracellular vesicles (EVs) demonstrate growing potential as innovative cell‐derived nanobiotherapies in diverse medical contexts, their physical properties (size, integrity, transport, etc.) in drug product formulation remain a critical concern poorly addressed so far. Herein, a methodology that relies on nanoparticle tracking analysis by interferometric light microscopy (ILM) for analyzing the concentration and size distribution of nanoparticles as well as their interactions with their local environment through a nanorheological approach is introduced. The analysis of interference patterns enables nanoparticles tracking not only in aqueous solutions but also in complex media with high‐viscosity or non‐Newtonian behavior, particularly pertinent for characterizing EV formulations. A proof of concept for in situ tracking of EVs suspended in Poloxamer‐407 as drug delivery system is presented. The ILM‐based analysis enables to 1) measure the viscosity at the nanoscale for Newtonian and non‐Newtonian fluids via calibration beads; 2) analyze data to determine the size distribution of EVs in non‐Newtonian complex fluid such as poloxamer formulation, and 3) analyze the interactions of EVs with poloxamer‐407. The proposed approach represents a valuable tool to understand the nanorheological behavior of EVs in viscoelastic media in situ as well as a quality control test for EV formulations intended to clinical use.