Extracellular vesicles (EVs), specifically exosomes of 50–150 nm, have emerged as important communication channels between cells and tissues and can be isolated from multiple biofluids including blood, urine and amniotic fluid. No standardized approach for exosome isolation from these biofluids has been established. This chapter outlines an optimized approach for isolating exosomes from human amniotic fluid samples. Like plasma, amniotic fluid contains many protein and cellular contaminants that requires multiple steps for cleanup. Therefore, to ensure samples contain minimal contaminants, including larger EVs, we also outline multiple methods for characterization of isolated exosomes for size, morphology and protein markers.