Extracellular vesicles (EVs) are cell-derived vesicles which encapsulate a variety of molecules. Numerous studies have demonstrated EVs as signaling mediators of intercellular communication and are heavily involved under physiological and pathological conditions. In translational medicine, EVs have been used for disease diagnosis and treatment monitoring. EVs as natural nanocarriers for drug delivery and therapeutic EVs are also under intense investigation. While still in its infancy, relevant EV studies have been growing. For EV-centered research to thrive, a few fundamental unanswered questions, such as EV biogenesis, EV secretion rate (SR), EV content sorting mechanisms, etc. require further investigation. In this study, we measured the SR of EVs derived from 6 cancerous cell lines. Several factors that may interfere with EV secretion, isolation, and storage were also investigated. Our results show that the SR of EVs derived from various cancer cells was significantly different, indicating a heterogeneous EV secretion behavior among cell types. Moreover, 5 different drugs that interfere with cellular metabolism significantly influenced EV release. In addition, we found that (1) more EVs can be harvested at 24 h compared to 48 h of serum-free cell culture with a similar degree of FBS contamination; (2) filtration of the cell culture supernatant with a 0.22 μm filter causes ∼70% loss of EVs; (3) the isolation efficiency of EVs with the prevalent ultracentrifugation is only ∼14%; (4) storage at 4 °C for 3 days causes ∼21% loss of EVs. Overall, our findings provide a guideline for proper EV collection and storage in laboratory settings.