Extracellular vesicles (EVs) are membrane vesicles secreted by cells and distributed widely in all biofluids. EVs can modulate the biological activities of cells in a paracrine or endocrine manner, in part by transferring their content, such as miRNA, following uptake in recipient cells. Fluorescent labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. Lipophilic fluorescent dyes such as those in the PKH family have been widely used for EV labelling. One concern with the use of lipophilic dyes is an increase in the EV size. This size shift alone may undermine the validity of EVs tracing studies as small changes in the size of inorganic nanoparticles are known to affect their cellular uptake and biodistribution. Here, the possibility of minimizing the size shift of PKH labelled EVs was systematically studied by changing the labelling condition. Unfortunately, the size shift towards larger particles was observed in all the PKH labelling conditions, including those where the labelled EVs were below the fluorescent detection limit. As opposed to lipophilic dyes, no significant shift in the size of labelled EVs was detected with protein binding dyes. Since the size shifts identified in all the PKH labelling conditions are likely to affect the cellular uptake and biodistribution, PKH may not be a reliable technique for EVs tracking.