Advances in high-throughput screening, together with rapid progress in genomic and proteomic sciences, have considerably stimulated the development of a variety of biomarker discovery tools and have contributed to the improvement of clinical diagnosis. Major challenges include the obtaining of high-quality data sets based on assays that are rapid, reliable, and inexpensive, but still highly sensitive and easy to perform. The microsphere-based flow cytometric technology is a platform that is currently entering the diagnostic market. This article reviews the development and the applications of microsphere-based flow cytometric platforms in basic and applied proteomic research, and discusses bead-based assays used in clinical applications.