Exosomes are cell-derived vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a non-invasive reservoir for biomarker discovery. Current methods for identifying and quantifying human urinary exosomes are time-consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) is a technology that can size and count nanoparticles, such as those released from cultured cells and in human plasma. NTA is based on the principle that at any particular temperature, the rate of Brownian motion of nanoparticles in solution is determined solely by their size. Published studies demonstrate that NTA can count and size specific sub-groups of particles using fluorescent antibodies against surface proteins, but this has not yet been applied to urine. In the current study we applied NTA to human urine (n=5) and identified a range of nanoparticles, including exosomes. Using antibodies against the exosomal marker proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a sub-population of CD24 and AQP2 positive particles of characteristic exosomal size (figure 1). Extensive pre-NTA processing of urine was not necessary; however, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultra-centrifugation step preceded NTA. Building upon previous work in our group in investigating exosomal signalling between cells, we were also able to track exosomal AQP2 upregulation induced by desmopressin stimulation of murine collecting duct cells by using NTA. Finally, when urine was stored at room temperature, 4C or frozen, nanoparticle concentration was reduced; freezing at -80C produced the least reduction. This reduction was substantially reduced by addition of protease inhibitors to urine before storage (figure 2). In conclusion, with appropriate sample storage, NTA has potential as a tool for rapidly characterising and quantifying exosome concentration in human urine and for further understanding the mechanism of intra-cellular signalling mediated by exosomes.