Understanding a molecular basis for cellular function is a common goal of biomedicine. The complex and dynamic cellular processes underlying physiological processes become subtly or grossly perturbed in human disease. A primary objective is to demystify this complexity by creating and establishing relevant model systems to study important aspects of human disease. Although significant technological advancements over the last decade in both genomic and proteomic arenas have enabled progress, accessing the complexity of cellular interactions that occur in vivo has been a difficult arena in which to make progress. Moreover, there are extensive challenges in translating research tools to clinical applications. Flow cytometry, over the course of the last 40 years, has revolutionized the field of immunology, in both the basic science and clinical settings, as well as having been instrumental to new and exciting areas of discovery such as stem cell biology. Multiparameter machinery and systems exist now to access the heterogeneity of cellular subsets and enable phenotypic characterization and functional assays to be performed on material from both animal models and humans. This review focuses primarily on the development and application of using activation-state readouts of intracellular activity for phospho-epitopes. We present recent work on how a flow cytometric platform is used to obtain mechanistic insight into cellular processes as well as highlight the clinical applications that our laboratory has explored. Furthermore, this review discusses the challenges faced with processing high-content multidimensional and multivariate data sets. Flow cytometry, as a platform that is well situated in both the research and clinical settings, can contribute to drug discovery as well as having utility for both biomarker and patient-stratification.