Robust and well-established orthogonal techniques for characterization of individual extracellular vesicles (EVs) are required to utilize them as therapeutic and diagnostic tools. However, to better utilize any analytical technology, we first need to determine the limitations of that technology. We have established capabilities to reliably analyze EVs samples using three different orthogonal techniques; nanoparticle tracking analysis, flow cytometry and analytical HPLC. Using these three platforms, EV samples can be analyzed reliably and in agreement with MISEV and MIFlowCyt-EV guidelines. Scattering and fluorescence mode of NTA suffer from masking effect and photobleaching respectively. We have optimized the labeling and measurement parameters to achieve reliable measurements. We will provide guidelines on how to characterize EVs accurately using any single particle analysis technique. We tested EV standards from Sigma (tested at ISEVxTech 2022) and measured identical particle concentration on 3 orthogonal techniques sc-NTA, fl-NTA and flow cytometry. We compared the size distribution using 5 techniques, sc-NTA (static), sc-NTA (flow), fl-NTA (flow), DLS and Cryo-EM. We found out that fl-NTA (flow) and Cryo-EM (as the gold standard) agreed the most with each other. Different nanoparticle families such as liposomes, lipid nanoparticles, mammalian EVs, and bacterial EVs were successfully characterized using this analytical toolbox. Single particle analysis techniques are less reliable when crude EV samples are tested. We used BSA as a model of non-EV associated protein contaminants. We observed that the free protein concentration must be below 50 µg/mL for scattering and 5 µg/mL for fluorescence NTA to characterize EVs accurately. Therefore, the relevant ratio of EVs compared to other non-EV components is a critical parameter in reliably analyzing crude EV samples. MSC conditioned media can be successfully characterized throughout each step of the purification process including clarification, tangential flow filtration, and chromatography. The optimization of labeling and measurement parameters is a critical step to characterize EVs reliably. Here, We provide guidelines on performing systematic and fundamental studies using standard samples and models for both EVs and protein contaminations in the background for any analytical technology.