Background <p>Small extracellular vesicles (sEVs) or exosomes are small-sized (30–150&#xa0;nm), nanoparticles that are released from nearly all cells under normal and pathophysiological conditions. The sEVs have a vital role in biological systems as they communicate and transfer their contents, such as proteins, lipids, and nucleic acids, from the cells of origin to nearby or distant cells. There is a growing interest in sEVs due to their potential applications in understanding disease mechanisms, identifying biomarkers, making clinical diagnoses, and developing therapeutics. Human biofluids such as blood, saliva, and urine are routinely collected in clinical settings and hold significant potential for isolating sEVs for accurate and early clinical biomarker identification. Currently, there is no established method for isolating sEVs in a single method utilizing different human biofluids. Here, we present a protocol for isolating sEVs from small volumes of plasma, saliva, and urine, that are clinically relevant for identifying biomarkers and for subsequent downstream applications.</p> Methods <p>Our method is specifically designed to efficiently isolate sEVs from 1 mL of blood, 2 mL of saliva, and 10 mL of urine samples using a widely available ultracentrifugation instrument. We examined the morphology of sEVs using Transmission Electron Microscopy and Scanning Electron Microscopy. The size distribution and concentration were determined using Nanoparticle Tracking Analysis. Additionally, we assessed exosome-specific markers such as CD-9, CD-63, Flotillin 1, and TSG 101 using western blotting. Our method has undergone successful testing for high-throughput applications with small sample volumes, demonstrating its clinical utility.</p> Results <p>We compared and discussed the results obtained from our tested method for isolating sEVs from three different sample sources. We used sequential centrifugation combined with filtration and sucrose gradient ultracentrifugation. We compared the yields of sEVs from these samples and found that this method resulted in high purity and yield of EVs. Further, we have identified the isolated proteins using mass spectrometry.</p> Discussion <p>The ease of obtaining patient samples from biofluids such as plasma, saliva, and urine makes them valuable for diagnostic purposes. The isolation of sEVs allows for the early diagnosis and prediction of diseases. Our method is simple to use, cost-effective, and reproducible. It has been tested with small sample volumes using proteomics and RNA analysis. When applied to human biofluids, this method can identify clinical research biomarkers, understand disease mechanisms, and monitor disease progression, among other related applications.</p>