Extracellular vesicles (EVs) are endogenic nanovesicles found in all biological fluids in the body, including that in the early stage of cancer development. This makes EVs as an exciting window into the body that could be utilised for the early detection of cancer. At the first step in establishing the method for detecting and quantifying EpCAM-positive EVs, in this work, we investigated EVs production from human colorectal adenocarcinoma cells-HT29 cells (EpCAM-positive cells) and normal human kidney cells-HEK 293T cells (EpCAM-negative cells) over different media containing various amount of exosome-depleted FBS (EDS). The cells at 70% of confluence were incubated with such media for 48 hours before cell culture media (CCM) being processed through serial centrifugations and characterized with size distribution and EVs concentration using Nanoparticle Tracking Analysis. A number of live cells after CCM collection also was determined for further calculations. We observed that a number of EVs produced from one HEK 293T cell was 10-fold higher than the figure for one HT29 cell. Moreover, while the concentrations of EDS-derived EVs increased against the growth of EDS amount in media, the total EV concentrations after incubating cells with the media remained similar for both cell lines. These findings suggest that HT29 cells and HEK 293T cells likely uptook EDS-derived EVs in the media, then regulated total EV concentration in CCM. With further studies, these results may contribute to understanding the way cells uptake serum-derived EVs and the effect of serum-derived EVs in CCM on the secretion of interested EVs by cells (including the regulation of EVs concentrations in CCM).