Flow cytometric, microsphere-based immunoassays have been developed for the simultaneous detection of soluble analytes in a variety of sample types. The ability to discriminate between individual microspheres on the basis of size, fluorescent intensity, and/or wavelength has allowed the simultaneous analysis of multiple analytes from the same sample. Cytokines are particularly good candidates for multiplexed analysis. Through intricate networks and complex feedback mechanisms, cytokines modulate each other as well as a multitude of cellular events and play an important role in health and disease. The simultaneous measurement of multiple cytokines in a single biological sample has tremendous potential value to further our understanding of the role of these immunomodulators in health and disease. This chapter describes a multiplexed, microsphere-based flow cytometric method to quantitate and compare multiple cytokines simultaneously in human serum. Serum poses a challenge for multiplexed cytokine analysis owing to the presence of numerous proteins and other potentially interfering factors. Assessment of the “real world” performance of the multiplexed, microsphere-based flow cytometry assay with clinically relevant sample types is critical for determining the proper application of these methods in deciphering the role of cytokines in disease pathogenesis and for evaluating drug action.