In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110000 x g or 40000 x g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5x centrifugation enrichment was found to provide similar or better performance than 5x UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5x UCs and 5x centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.